Double-Acylated GLP-1 Derivatives

ABSTRACT

The invention also relates to the pharmaceutical use thereof, for example in the treatment and/or prevention of all forms of diabetes and related diseases, as well as to corresponding novel peptide and linker intermediates. The derivatives are potent, stable, protracted, and suitable for oral administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/966,642, filed Apr. 30, 2018, which is a continuation of U.S.application Ser. No. 14/399,087, filed Nov. 5, 2014 (now U.S. Pat. No.10,000,542, issued Jun. 19, 2018), which is a 35 U.S.C. § 371 NationalStage application of International Application PCT/EP2013/059112(WO2013/167454), filed May 2, 2013, which claimed priority of EuropeanPatent Application 12167091.3, filed May 8, 2012 and European PatentApplication 12183246.3, filed Sep. 6, 2012; and of U.S. ProvisionalApplication 61/646,470; filed May 14, 2012 and U.S. ProvisionalApplication 61/741,770; filed Sep. 6, 2012; the contents of which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to derivatives of analogues ofGlucagon-Like Peptide 1 (GLP-1), more in particular to double-acylatedGLP-1 derivatives acylated at K²⁶ and at K³⁷, and their pharmaceuticaluse.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 4, 2019, isnamed 8503US03_SeqList.txt and is 7 kilobytes in size.

BACKGROUND

-   Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-669    discloses derivatives of GLP-1(7-37) that are double-acylated at    K^(26,34).-   WO 2011/080103 A1 discloses a number of double-acylated GLP-1    derivatives acylated at K²⁶ and at K³⁷.-   WO 99/43706 A1 discloses a number of mono- and double-acylated GLP-1    derivatives.-   WO 98/08871 A1 discloses a number of GLP-1 derivatives including    some that are double-acylated.

SUMMARY

The invention relates to derivatives of GLP-1 peptides.

Liraglutide is a mono-acylated GLP-1 derivative for once dailyadministration which is marketed as of 2009 by Novo Nordisk A/S. Thiscompound is disclosed in WO 98/08871 A1 (Example 37).

WO 06/097537 A2 discloses among other GLP-1 derivatives semaglutide(Example 4) which is a mono-acylated GLP-1 derivative for once weeklyadministration which is under development by Novo Nordisk A/S.

The derivatives of the invention are acylated at a lysine substitutedfor the native glycine at position 37, as well as at the native lysineat position 26. The side chains are albumin binding moieties. Theycomprise a protracting moiety, preferably selected from fatty diacids,and fatty acids with a distal phenoxy group, all optionally substituted.A carboxy group of the fatty acid or fatty diacid is acylated,optionally via a linker, to a lysine residue of the GLP-1 peptide,preferably at the epsilon-amino group thereof.

The GLP-1 peptide may be an analogue of GLP-1 (7-37) (SEQ ID NO: 1)having a total of up to eight amino acid differences as compared toGLP-1(7-37), for example one or more additions, one or more deletions,and/or one or more substitutions.

More in particular, the invention relates to a derivative of a GLP-1analogue, which analogue comprises a first K residue at a positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 37 of GLP-1(7-37) (SEQID NO: 1), and a maximum of eight amino acid changes as compared toGLP-1(7-37), which derivative comprises two protracting moietiesattached to said first and second K residue, respectively, each via alinker, wherein the protracting moiety is selected from Chem. 1:HOOC—(CH₂)_(x)—CO—*, and Chem. 2: HOOC—C₆H₄—O—(CH₂)_(y)—CO—*, in which xis an integer in the range of 8-16, and y is an integer in the range of6-13; and each linker comprises Chem. 3a:*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NR₁R₂]—CO—*, which is connected at its CO—*end to the epsilon amino group of the first or the second K residue ofthe GLP-1 analogue, and wherein q is an integer in the range of 0-5, R₁and R₂ independently represent a hydrogen radical (*—H) or methyl(*—CH₃), and w is an integer in the range of 0-5; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

The invention also relates to such derivative for use as a medicament,in particular for use in the treatment and/or prevention of all forms ofdiabetes and related diseases, such as eating disorders, cardiovasculardiseases, gastrointestinal diseases, diabetic complications, criticalillness, and/or polycystic ovary syndrome; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression.

The invention furthermore relates to intermediate products in the formof novel GLP-1 analogues, which are relevant for the preparation ofcertain derivatives of the invention.

The invention furthermore relates to an intermediate compound in theform of a novel protected compound (Chem. 63, Example 43) that may beused in the synthesis of the compounds of Examples 29 and 37 herein andsimilar compounds.

The derivatives of the invention are biologically active. For example,they are much more potent and bind much better to the GLP-1 receptor ascompared to their respective direct comparator compound which differsonly in the linker. Also, or alternatively, they have a protractedpharmacokinetic profile. Also, or alternatively, they have a high oralbioavailability. These properties are of importance in the developmentof next generation GLP-1 compounds for subcutaneous, intravenous, and/orin particular oral administration.

DESCRIPTION

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ε=epsilon;γ=gamma; ω=omega; etc. Also, the Greek letter of g may be represented by“u”, e.g. in μl=ul, or in μM=uM.

An asterisk (*) in a chemical formula designates i) a point ofattachment, ii) a radical, and/or iii) an unshared electron.

In a first aspect, the invention relates to a derivative of a GLP-1analogue, which analogue comprises a first K residue at a positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 37 of GLP-1(7-37) (SEQID NO: 1), and a maximum of eight amino acid changes as compared toGLP-1(7-37), which derivative comprises two protracting moietiesattached to said first and second K residue, respectively, via a linker,wherein the protracting moiety is selected from Chem. 2, and Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

HOOC—(CH₂)_(x)—CO—*,  Chem. 1:

in which x is an integer in the range of 8-16, and y is an integer inthe range of 6-13; and the linker comprises

*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NR₁R₂]—CO—*,  Chem. 3a:

which is connected at its CO—* end to the epsilon amino group of thefirst or the second K residue of the GLP-1 analogue, and wherein q is aninteger in the range of 0-5, R₁ and R₂ independently represent *—H or*—CH₃, and w is an integer in the range of 0-5; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

GLP-1 Analogues

The term “GLP-1 analogue” or “analogue of GLP-1” as used herein refersto a peptide, or a compound, which is a variant of the humanGlucagon-Like Peptide-1 (GLP-1 (7-37)), the sequence of which isincluded in the sequence listing as SEQ ID NO: 1. The peptide having thesequence of SEQ ID NO: 1 may also be designated “native” GLP-1.

In the sequence listing, the first amino acid residue of SEQ ID NO: 1(histidine) is assigned no. 1. However, in what follows—according toestablished practice in the art—this histidine residue is referred to asno. 7, and subsequent amino acid residues are numbered accordingly,ending with glycine no. 37. Therefore, generally, any reference hereinto an amino acid residue number or a position number of the GLP-1 (7-37)sequence is to the sequence starting with His at position 7 and endingwith Gly at position 37.

GLP-1 analogues of the derivatives of the invention may be described byreference to i) the number of the amino acid residue in native GLP-1(7-37) which corresponds to the amino acid residue which is changed(i.e., the corresponding position in native GLP-1), and to ii) theactual change. The following are non-limiting examples of suitableanalogue nomenclature.

A non-limiting example of a GLP-1 analogue of the derivative of theinvention is an analogue that is changed so as to comprise a lysineresidue at a position corresponding to position 37 of GLP-1(7-37). AGLP-1 analogue of the derivative of the invention also comprises alysine residue at a position corresponding to position 26 (the nativelysine). The amino acid sequence of this analogue may be otherwiseidentical to that of native GLP-1, and this analogue may accordingly bedesignated K³⁷-GLP-1 (7-37). This designation represents the amino acidsequence of native GLP-1 where glycine at position 37 has beensubstituted with lysine.

The GLP-1 analogue forming part of the derivative of the inventioncomprises a maximum of eight amino acid changes when compared withnative GLP-1(7-37) (SEQ ID NO: 1). In other words, it is a GLP-1(7-37)peptide in which a number of amino acid residues have been changed whencompared to native GLP-1(7-37) (SEQ ID NO: 1). These changes mayrepresent, independently, one or more amino acid substitutions,additions, and/or deletions.

The following are non-limiting examples of appropriate analoguenomenclature.

For example, the analogue [Aib⁸, Glu²², Gln³⁴,Lys³⁷]-GLP-1-(7-37)designates a GLP-1 (7-37) peptide which, when compared to native GLP-1,has the following substitutions: Substitution of alanine at position 8with Aib (α-aminoisobutyric acid), of glycine at position 22 withglutamic acid, of lysine at position 34 with glutamine, and of glycineat position 37 with lysine. This analogue may also be briefly designated(8Aib, 22E, 34Q, 37K), where reference to GLP-1 (7-37) is implied.

Analogues “comprising” certain specified changes may comprise furtherchanges, when compared to SEQ ID NO: 1.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent.

The expressions “a position equivalent to” or “corresponding position”may be used to characterise the site of change in a variant GLP-1 (7-37)sequence by reference to native GLP-1(7-37) (SEQ ID NO: 1). Equivalentor corresponding positions, as well as the number of changes, are easilydeduced, e.g. by simple handwriting and eyeballing; and/or a standardprotein or peptide alignment program may be used, such as “align” whichis a Needleman-Wunsch alignment. The algorithm is described inNeedleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48: 443-453, and the align program by Myers and W. Miller in“Optimal Alignments in Linear Space” CABIOS (computer applications inthe biosciences) (1988) 4:11-17. For the alignment, the default scoringmatrix BLOSUM50 and the default identity matrix may be used, and thepenalty for the first residue in a gap may be set at −12, or preferablyat −10, and the penalties for additional residues in a gap at −2, orpreferably at −0.5.

An example of such alignment is inserted hereinbelow, in which sequenceno. 1 (SEQ_ID_NO_1) is SEQ ID NO: 1, and sequence no. 2 (SEQ ID NO: 2)(ANALOGUE) is the analogue (8Aib, 22E, 34Q, 37K) thereof:

# Aligned_sequences: 2 #1: SEQ_ID_NO_1 #2: ANALOGUE # Matrix: EBLOSUM62# Gap_penalty: 10.0 # Extend_penalty: 0.5

#

# Length: 31 # Identity: 27/31 (87.1%) # Similarity: 28/31 (90.3%) #Gaps: 0/31 (0.0%) # Score: 137.0

SEQ_ID_NO_1 1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 31  |.|||||||||||||.|||||||||||:||. ANALOGUE (SEQ ID NO: 2)1 HxEGTFTSDVSSYLEEQAAKEFIAWLVQGRK 31

As can be inferred from the above, in case of non-natural amino acidssuch as Aib being included in the sequence, these may, for alignmentpurposes, be replaced with X. If desired, X can later be manuallycorrected.

The term “peptide”, as e.g. used in the context of the GLP-1 analoguesof the derivatives of the invention, refers to a compound whichcomprises a series of amino acids intereconnected by amide (or peptide)bonds.

The peptides of the invention comprise at least five constituent aminoacids connected by peptide bonds. In particular embodiments the peptidecomprises at least 10, preferably at least 15, more preferably at least20, even more preferably at least 25, or most preferably at least 28amino acids.

In particular embodiments, the peptide is composed of at least fiveconstituent amino acids, preferably composed of at least 10, at least15, at least 20, at least 25, or most preferably composed of at least 28amino acids.

In additional particular embodiments, the peptide is a) composed of, orb) consists of, i) 28, ii) 29, iii) 30, iv) 31, v) 32, or vi) 33 aminoacids.

In a still further particular embodiment the peptide consists of aminoacids interconnected by peptide bonds.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes proteogenic amino acids (encoded by thegenetic code, including natural amino acids, and standard amino acids),as well as non-proteogenic (not found in proteins, and/or not coded forin the standard genetic code), and synthetic amino acids. Thus, theamino acids may be selected from the group of proteinogenic amino acids,non-proteinogenic amino acids, and/or synthetic amino acids.

Non-limiting examples of amino acids which are not encoded by thegenetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.Non-limiting examples of synthetic amino acids are the D-isomers of theamino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyricacid), β-alanine, and des-amino-histidine (desH, alternative nameimidazopropionic acid, abbreviated Imp).

In what follows, all specific amino acids for which the optical isomeris not stated is to be understood to mean the L-isomer (unless otherwisespecified), e.g. when reference is made to the specific amino acid ofglutamine, this is intended to refer to L-glutamine, unless otherwise isstated. On the other hand, where amino acids are described by moregeneral formulas such as brutto formulas or structural formulas and whenno stereo chemistry is shown, these formulas are intended to cover allstereo isomers.

The GLP-1 derivatives and analogues of the invention have GLP-1activity. This term refers to the ability to bind to the GLP-1 receptorand initiate a signal transduction pathway resulting in insulinotropicaction or other physiological effects as is known in the art. Forexample, the analogues and derivatives of the invention can be testedfor GLP-1 activity using the assays described in Examples 44 and 45herein. The GLP-1 receptor binding assay described in Example 46 hereinmay also, if relevant, be used as a measure of GLP-1 activity, or, moreprecisely, GLP-1 receptor affinity (the low HSA experiment).

GLP-1 Derivatives

The term “derivative” as used herein in the context of a GLP-1 peptideor analogue means a chemically modified GLP-1 peptide or analogue, inwhich one or more substituents have been covalently attached to thepeptide. The substituent may also be referred to as a side chain.

In a particular embodiment, the side chain is capable of formingnon-covalent aggregates with albumin, thereby promoting the circulationof the derivative with the blood stream, and also having the effect ofprotracting the time of action of the derivative, due to the fact thatthe aggregate of the GLP-1-derivative and albumin is only slowlydisintegrated to release the active pharmaceutical ingredient. Thus, thesubstituent, or side chain, as a whole is preferably referred to as analbumin binding moiety.

In another particular embodiment the albumin binding moiety comprises aportion which is particularly relevant for the albumin binding andthereby the protraction, which portion may be referred to as aprotracting moiety. The protracting moiety may be at, or near, theopposite end of the albumin binding moiety, relative to its point ofattachment to the peptide.

In a still further particular embodiment the albumin binding moietycomprises a portion inbetween the protracting moiety and the point ofattachment to the peptide, which portion may be referred to as a linker,linker moiety, spacer, or the like. The linker may be optional, andhence in that case the albumin binding moiety may be identical to theprotracting moiety.

In particular embodiments, the albumin binding moiety and/or theprotracting moiety is lipophilic, and/or negatively charged atphysiological pH (7.4).

The albumin binding moiety, the protracting moiety, or the linker may becovalently attached to a lysine residue of the GLP-1 peptide byacylation.

In a preferred embodiment, an active ester of the albumin bindingmoiety, preferably comprising a protracting moiety and a linker, iscovalently linked to an amino group of a lysine residue, preferably theepsilon amino group thereof, under formation of an amide bond (thisprocess being referred to as acylation).

Unless otherwise stated, when reference is made to an acylation of alysine residue, it is understood to be to the epsilon-amino groupthereof.

A derivative comprising two protracting moieties attached to a first anda second K residue (viz. to K²⁶ and K³⁷) via a linker may be referred toas a derivative which has been acylated twice, double-acylated, or dualacylated at the epsilon-amino groups of the first and second lysineresidues, e.g. at position 26 and 37, respectively, of the GLP-1peptide.

For the present purposes, the terms “albumin binding moiety”,“protracting moiety”, and “linker” may include the unreacted as well asthe reacted forms of these molecules. Whether or not one or the otherform is meant is clear from the context in which the term is used.

In one aspect, each protracting moiety comprises, or consists of, aprotracting moiety independently selected from Chem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 8-16, and y is an integer inthe range of 6-13.

-   -   In one embodiment, *—(CH₂)_(x)—* refers to straight alkylene in        which x is an integer in the range of 8-16.

In another embodiment, *—(CH₂)_(y)—* refers to straight alkylene inwhich y is an integer in the range of 6-13.

The term “fatty acid” refers to aliphatic monocarboxylic acids havingfrom 4 to 28 carbon atoms, it is preferably unbranched, and/or evennumbered, and it may be saturated or unsaturated.

The term “fatty diacid” refers to fatty acids as defined above but withan additional carboxylic acid group in the omega position. Thus, fattydiacids are dicarboxylic acids.

The nomenclature is as is usual in the art, for example in the aboveformulas *—COOH as well as HOOC—* refers to carboxy; *—C₆H₄—* tophenylene; *—CO—*, as well as *—OC—*, to carbonyl (O═C<**); C₆H₅—O—* tophenoxy. Furthermore, CO—* refers to C(═O)—*. For example, in anyformula (R—CO—*) herein (where R is as defined by each formula), R—CO—*,refers to R—C(═O)—*. In particular embodiments, the aromatics, such asthe phenoxy, and the phenylene radicals, may be para.

As explained above, the GLP-1 derivatives of the present invention aredouble-acylated, i.e. two albumin binding moieties are covalentlyattached to the GLP-1 peptide.

In a particular embodiment, the two albumin binding moieties (i.e. theentire side chains) are similar, preferably substantially identical, or,most preferably, identical.

In another particular embodiment, the two protracting moieties aresimilar, preferably substantially identical, or, most preferably,identical.

In a still further particular embodiment, the two linkers are similar,preferably substantially identical, or, most preferably identical.

The term “substantially identical” includes differences from identitywhich are due to formation of one or more salts, esters, and/or amides;preferably formation of one or more salts, methyl esters, and simpleamides; more preferably formation of no more than two salts, methylesters, and/or simple amides; even more preferably formation of no morethan one salt, methyl ester, and/or simple amide; or most preferablyformation of no more than one salt.

In the context of chemical compounds such as the albumin bindingmoieties, protracting moieties, and linkers, similarity and/or identitymay be determined using any suitable computer program and/or algorithmknown in the art.

For example, the similarity of two protracting moieties, two linkers,and/or two entire side chains may suitably be determined using molecularfingerprints. Fingerprints is a mathematical method of representing achemical structure (see e.g. Chemoinformatics: A textbook, JohannGasteiger and Thomas Engel (Eds), Wiley-VCH Verlag, 2003).

Examples of suitable fingerprints include, without limitation, UNITYfingerprints, MDL fingerprints, and/or ECFP fingerprints, such as ECFP_6fingerprints (ECFP stands for extended-connectivity fingerprints).

In particular embodiments, the two protracting moieties, the twolinkers, and/or the two entire side chains are represented as a) ECFP_6fingerprints; b) UNITY fingerprints; and/or c) MDL fingerprints.

The Tanimoto coefficient is preferably used for calculating thesimilarity of the two fingerprints, whether a), b), or c) is used.

In particular embodiments, whether a), b), or c) is used, the twoprotracting moieties, the two linkers, and/or the two entire sidechains, respectively, have a similarity of at least 0.5 (50%);preferably at least 0.6 (60%); more preferably at least 0.7 (70%), or atleast 0.8 (80%); even more preferably at least 0.9 (90%); or mostpreferably at least 0.99 (99%), such as a similarity of 1.0 (100%).

UNITY fingerprints may be calculated using the programme SYBYL(available from Tripos, 1699 South Hanley Road, St. Louis, Mo.63144-2319 USA). ECFP_6 and MDL fingerprints may be calculated using theprogramme Pipeline Pilot (available from Accelrys Inc., 10188 TelesisCourt, Suite 100, San Diego, Calif. 92121, USA).

For more details, see for example J. Chem. Inf. Model. 2008, 48,542-549; J. Chem. Inf. Comput. Sci. 2004, 44, 170-178; J. Med. Chem.2004, 47, 2743-2749; J. Chem. Inf. Model. 2010, 50, 742-754; as well asSciTegic Pipeline Pilot Chemistry Collection: Basic Chemistry UserGuide, March 2008, SciTegic Pipeline Pilot Data Modeling Collection,2008—both from Accelrys Software Inc., San Diego, US, and the guideshttp://www.tripos.com/tripos_resources/fileroot/pdfs/Unity_111408.pdf,and http://www.tripos.com/data/SYBYL/SYBYL_072505.pdf.

An example of a similarity calculation is inserted hereinbelow, in whicha known entire side chain of a known GLP-1 derivative was compared witha methyl ester thereof:

Using a) ECFP_6 fingerprints the similarity is 0.798, using b) UNITYfingerprints the similarity is 0.957; and using MDL fingerprints thesimilarity is 0.905.

In case of two identical side chains (albumin binding moieties) thederivative may be designated symmetrical.

In particular embodiments, the similarity coefficient is at least 0.80,preferably at least 0.85, more preferably at least 0.90, even morepreferably at least 0.95, or most preferably at least 0.99.

Each of the two linkers of the derivative of the invention comprises thefollowing first linker element (A): Chem. 3a:*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NR₁R₂]—CO—*, which is connected at its CO—*end to the epsilon amino group of the first or the second K residue ofthe GLP-1 analogue, wherein q is an integer in the range of 0-5, R₁ andR₂ independently represent *—H (a hydrogen radical), or *—CH₃ (methyl),and w is an integer in the range of 0-5.

One non-limiting example of a linker comprising this first linkerelement of Chem. 3a is lysine, or rather a di-radical of lysine. Lysinemay preferably be used as a linker in its omega-version, where omegarefers to the fact that it is the amino group at the distal C-atom ofthe alkyl substituent chain that is radicalised (to *—NH). For lysinethe omega position is herein generally referred to as the epsilonposition (the alpha atom is the C-atom next to the carboxylic acidfunction, and each upstream C-atom is then designated using thesubsequent Greek letters beta, gamma, delta, and so on, until the C-atomto which the *—NH group is attached is reached, and it is in case oflysine the epsilon atom). Accordingly, when w=0, R₁ and R₂ bothrepresent *—H, and q=4, the formula Chem. 3a refers to a di-radical ofepsilon-lysine (eps-Lys; Chem. 6).

Another non-limiting example of a linker comprising this first linkerelement of Chem. 3a is N^(α),N^(α)-dimethyl lysine, or rather adi-radical of this residue (6-amino-(S)-2-(dimethylamino)hexanoyl). AlsoN^(α),N^(α)-dimethyl lysine may preferably be used as a linker in itsomega-version, where omega refers to the fact that it is the amino groupat the distal C-atom of the alkyl substituent chain that is radicalised(to *—NH). Also for N^(α),N^(α)-dimethyl lysine the omega position isherein generally referred to as the epsilon position (the alpha atom isthe C-atom next to the carboxylic acid function, and each upstreamC-atom is then designated using the subsequent Greek letters beta,gamma, delta, and so on, until the C-atom to which the *—NH group isattached is reached, and it is in case of N^(α),N^(α)-dimethyl lysinethe epsilon atom). Accordingly, when w=0, R, and R₂ both represent*—CH₃, and q=4, the formula Chem. 3a refers to a di-radical ofN^(α),N^(α)-dimethyl epsilon lysine (in brief “N,N-dimethyl-eps-Lys”;Chem. 6a).

In a preferred embodiment, this first linker element is in its L-form.

The linker may comprise 1 or 2 times Chem. 3a. When z is 2 the Chem. 3aelements are preferably interconnected via an amide bond. For example,the linker may comprise two times epsilon-Lys (2×eps-Lys; 2×Chem. 6).

The linker (each of the first and second linker) may further (i.e., inaddition to one or two times the first linker element (A)) comprise oneor more additional linker elements, e.g. linker elements independentlyselected from the second (B), third (C), fourth (D), fifth (E), and/orsixth (F) linker elements, as defined in the following:

A second linker element (B):

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.

In a particular embodiment, when k=1 and n=1, this linker element may bedesignated OEG, or 8-amino-3,6-dioxaoctanic acid, and/or it may berepresented by the following formula:

*—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—*.  Chem. 12a:

A third linker element (C), gamma-glutamic acid (gGlu):

In gamma-Glu (gGlu) it is the gamma carboxy group of the amino acidglutamic acid which is here used for connection to another linkerelement, or to the epsilon-amino group of lysine.

A fourth linker element (D), a di-radical of amino octanoic acid (Aoc):

*—NH—(CH₂)₇—CO—*.  Chem. 15:

A fifth linker element (E), being a di-radical of the amino acid serine(Ser).

A sixth linker element (F), being a di-radical of the amino acid glycine(Gly).

In one, non-limiting, particular embodiment, each linker consists ofChem. 14 and two times Chem. 6 (Chem.14-2×Chem.6), interconnected viaamide bonds and in the sequence indicated, connected at its *—NH end tothe CO—* end of the protracting moiety, and at its CO—* end to theepsilon amino group of the first or the second K residue of the GLP-1analogue.

For example, the first linker consists of (Chem.14-2×Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the first protracting moiety, and atits CO—* end to the epsilon amino group of the first K residue of theGLP-1 analogue; and the second linker consists of (Chem.14-2×Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the second protracting moiety, and atits CO—* end to the epsilon amino group of the second K residue of theGLP-1 analogue.

Needless to say, just for the sake of good order: Here and in thefollowing the phrase “in the sequence indicated” means that the *—NH endof the first-mentioned linker element (here Chem. 14) is connected tothe CO—* end of the protractor, and the CO—* end of the last-mentionedlinker element (here the last one of the two times Chem. 6) is connectedto the epsilon amino group of the K residue in question of the GLP-1analogue.

The derivatives of the invention may exist in different stereoisomericforms having the same molecular formula and sequence of bonded atoms,but differing only in the three-dimensional orientation of their atomsin space. The stereoisomerism of the examplified derivatives of theinvention is indicated in the experimental section, in the names as wellas the structures, using standard nomenclature. Unless otherwise statedthe invention relates to all stereoisomeric forms of the claimedderivative.

The concentration in plasma of the GLP-1 derivatives of the inventionmay be determined using any suitable method. For example, LC-MS (LiquidChromatography Mass Spectroscopy) may be used, or immunoassays such asRIA (Radio Immuno Assay), ELISA (Enzyme-Linked Immuno Sorbent Assay),and LOCI (Luminescence Oxygen Channeling Immunoasssay). Generalprotocols for suitable RIA and ELISA assays are found in, e.g.,WO09/030738 on p. 116-118. A preferred assay is the LOCI assay, e.g.described in Example 48 herein.

Intermediate Products

The invention also relates to an intermediate product in the form of aGLP-1 analogue which comprises the following changes as compared toGLP-1(7-37) (SEQ ID NO: 1): (ii) 8Aib, 34H, 37K; (v) 8Aib, 22E, 34Q,37K; (vii) 7Imp, 8Aib, 34R, 37K; (iix) 8Aib, 22E, 30E, 34R, 37K, 38E; or(ix) 8Aib, 22E, 30E, 34R, 37K; or a pharmaceutically acceptable salt,amide, or ester of any of the analogues of (ii), (v), (vii), (iix), or(ix).

The invention furthermore relates to an intermediate product in the formof a GLP-1 analogue selected from the following analogues of GLP-1(7-37) (SEQ ID NO: 1): (ii) 8Aib, 34H, 37K; (v) 8Aib, 22E, 34Q, 37K;(vi) 8Aib, 22E, 34R, 37K; (vii) 7Imp, 8Aib, 34R, 37K; (iix) 8Aib, 22E,30E, 34R, 37K, 38E; and (ix) 8Aib, 22E, 30E, 34R, 37K; or apharmaceutically acceptable salt, amide, or ester of any of theanalogues of (ii), (v), (vi), (vii), (iix), or (ix).

The invention furthermore relates to an intermediate product in the formof a novel protected linker compound that may be used in the synthesisof the compounds of Examples 29 and 37 herein, and similar compounds,namely the compound(S)-2-Dimethylamino-6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoicacid with the structure of Chem. 63:

or a pharmaceutically acceptable salt, amide, or ester thereof.

Pharmaceutically Acceptable Salt, Amide, or Ester

The intermediate products, analogues and derivatives of the inventionmay be in the form of a pharmaceutically acceptable salt, amide, orester.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.: 2 NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acid salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the derivatives of the invention may be formed with addedcations or anions that react with anionic or cationic groups,respectively. These groups may be situated in the peptide moiety, and/orin the side chain of the derivatives of the invention.

Non-limiting examples of anionic groups of the derivatives of theinvention include free carboxylic groups in the side chain, if any, aswell as in the peptide moiety. The peptide moiety often includes a freecarboxylic acid group at the C-terminus, and it may also include freecarboxylic groups at internal acid amino acid residues such as Asp andGlu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

The ester of the derivatives of the invention may, e.g., be formed bythe reaction of a free carboxylic acid group with an alcohol or aphenol, which leads to replacement of at least one hydroxyl group by analkoxy or aryloxy group

The ester formation may involve the free carboxylic group at theC-terminus of the peptide, and/or any free carboxylic group in the sidechain.

The amide of the derivatives of the invention may, e.g., be formed bythe reaction of an activated form of a free carboxylic acid group withan amine or a substituted amine, or by reaction of a free or substitutedamino group with an activated form of a carboxylic acid.

The amide formation may involve the free carboxylic group at theC-terminus of the peptide, any free carboxylic group in the side chain,the free amino group at the N-terminus of the peptide, and/or any freeor substituted amino group of the peptide in the peptide and/or the sidechain.

In a particular embodiment, the peptide or derivative is in the form ofa pharmaceutically acceptable salt. In another particular embodiment,the derivative is in the form of a pharmaceutically acceptable amide,preferably with an amide group at the C-terminus of the peptide. In astill further particular embodiment, the peptide or derivative is in theform a pharmaceutically acceptable ester.

Functional Properties

In a first aspect, the derivatives of the invention have a good potency.Also, or alternatively, in a second aspect, they have a protractedpharmacokinetic profile. Also, or alternatively, in a third aspect, theyhave a high oral bioavailability. Also, or alternatively, the number ofmutations in the GLP-1 analogue is low.

Biological Activity (Potency)

According to the first aspect, the derivatives of the invention, as wellas the constituent GLP-1 peptides as such (such as K³⁷-GLP-1(7-37) oranalogues thereof), are biologically active, or potent. For example, thederivatives of the invention are much more potent than their respectivedirect comparator compound which differs only in the linker.

More in particular, the Chem. 3a linker element has proven to be,surprisingly and unexpectedly, superior as compared to known linkerelements when it comes to biological activity or potency of GLP-1derivatives incorporating this linker element.

For example, as demonstrated in Example 50 herein, when a linkerconsisting of (Chem. 14-2×Chem.6), or (Chem.14-2×Chem.6a), is usedinstead of the “gGlu-2×OEG” (Chem. 14-2×Chem. 13) linker, which iswell-established in the art and recognised as an excellent linker, theGLP-1 derivatives of the present invention are, surprisingly andunexpectedly, much more potent as compared to these best-in-classstate-of-the-art GLP-1 derivatives.

In a particular embodiment, potency and/or activity refers to in vitropotency, i.e. performance in a functional GLP-1 receptor assay, more inparticular to the capability of activating the human GLP-1 receptor.

The in vitro potency may, e.g., be determined in a medium containingmembranes expressing the human GLP-1 receptor, and/or in an assay withwhole cells expressing the human GLP-1 receptor.

For example, purified plasma membranes from a stable transfected cellline expressing the human GLP-1 receptor may be stimulated with theGLP-1 analogue or derivative in question, and the potency of cAMPproduction measured, e.g. based on competition between endogenouslyformed cAMP and exogenously added biotin-labelled cAMP, which may becaptured using a specific antibody, e.g. as described in Example 44.

Also, or alternatively, the response of the human GLP-1 receptor may bemeasured in a reporter gene assay, e.g. in a stably transfected BHK cellline that expresses the human GLP-1 receptor and contains the DNA forthe cAMP response element (CRE) coupled to a promoter and the gene forfirefly luciferase (CRE luciferase). When cAMP is produced as a resultof activation of the GLP-1 receptor this in turn results in theluciferase being expressed. Luciferase may be determined by addingluciferin, which by the enzyme is converted to oxyluciferin and producesbioluminescence, which is measured and is a measure of the in vitropotency. One non-limiting example of such an assay is described inExample 45.

The term half maximal effective concentration (EC₅₀) generally refers tothe concentration which induces a response halfway between the baselineand maximum, by reference to the dose response curve. EC₅₀ is used as ameasure of the potency of a compound and represents the concentrationwhere 50% of its maximal effect is observed.

The in vitro potency of the derivatives of the invention may bedetermined as described above, and the EC₅₀ of the derivative inquestion determined. The lower the EC₅₀ value, the better the potency.

In a further particular embodiment, the derivative of the invention hasan in vitro potency corresponding to an EC₅₀ at or below 10000 pM, morepreferably below 5000 pM, even more preferably below 1000 pM, or mostpreferably below 500 pM (e.g. determined as described in Example 44.

In a still further particular embodiment, the derivative of theinvention has a potency corresponding to an EC₅₀ at 0% HSA of below 400pM, preferably below 300 pM, more preferably below 200 pM, even morepreferably below 150 pM, or most preferably below 100 pM (e.g.determined as described in Example 45.

Also, or alternatively, the ability of the derivatives of the inventionto bind to the GLP-1 receptor may be measured, and, if relevant, used asa measure of the GLP-1 activity (receptor affinity). This ability may bedetermined as described in Example 46. Generally, the binding to theGLP-1 receptor at low albumin concentration should be as good aspossible, corresponding to a low IC₅₀ value. In particular embodiments,the IC₅₀ value of a derivative of the invention, in the presence of0.001% HSA (low albumin), is below the corresponding IC₅₀ value forsemaglutide, preferably below 90% thereof, more preferably below 80%thereof, even more preferably below 70% thereof, or most preferablybelow 50% thereof.

For example, the derivatives of the invention are much more potent thantheir respective direct comparator compound which differs only in thelinker.

More in particular, the Chem. 3a linker element has proven to be,surprisingly and unexpectedly, superior as compared to known linkerelements when it comes to the binding to the GLP-1 receptor of GLP-1derivatives incorporating this linker element.

For example, as demonstrated in Example 50 herein, when a linkerconsisting of (Chem. 14-2×Chem.6), or (Chem.14-2×Chem.6a), is usedinstead of the “gGlu-2×OEG” (Chem. 14-2×Chem. 13) linker, which iswell-established in the art and recognised as an excellent linker, theGLP-1 derivatives of the present invention have, surprisingly andunexpectedly, a much better binding to the GLP-1 receptor as compared tothese best-in-class state-of-the-art GLP-1 derivatives.

In another particular embodiment the derivatives of the invention arepotent in vivo, which may be determined as is known in the art in anysuitable animal model, as well as in clinical trials.

The diabetic db/db mouse is one example of a suitable animal model, andthe blood glucose lowering effect, and/or the body weight loweringeffect may be determined in such mice in vivo, for body weight loweringeffect preferably after one acute administration.

The LYD pig is another example of a suitable animal model, and thereduction in food intake may be determined in a PD study in such pigs invivo, e.g. as described in Example 49. The derivatives of the inventionare very potent in vivo, which is evidenced by a nice reduction in foodintake in this PD study in pigs.

Protraction—Receptor Binding/Low and High Albumin

According to the second aspect, the derivatives of the invention areprotracted.

The ability of the derivatives of the invention to bind to the GLP-1receptor in the presence of a low and a high concentration of albumin,respectively, may be determined as described in Example 46.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their residence inplasma. Therefore, the IC₅₀ value at high albumin will generally behigher than the IC₅₀ value at low albumin, corresponding to a reducedbinding to the GLP-1 receptor, caused by albumin binding competing withthe binding to the GLP-1 receptor.

Protraction—Half Life In Vivo

According to the second aspect, the derivatives of the invention areprotracted.

Protraction may be determined as terminal half-life (T_(1/2)) in vivo inrats after i.v. administration, as described in Example 48. Inparticular embodiments, the half-life in rat is at least 10 hours,preferably at least 15 hours, more preferably at least 20 hours, evenmore preferably at least 25 hours, or most preferably at least 30 hours.

Or, protraction may be determined in another animal species, for exampleas terminal half-life (T_(1/2)) in vivo in minipigs after i.v.administration, as described in Example 47. In particular embodiments,the terminal half-life in minipigs is at least 8 hours, preferably atleast 24 hours, still more preferably at least 40 hours, even morepreferably at least 60 hours, or most preferably at least 80 hours.

Surprisingly, the present inventors identified a novel class of GLP-1derivatives, object of the present invention, which have a high potency,and at the same time preferably a long half-life.

Oral Bioavailability

According to the third aspect, the derivatives of the invention have ahigh oral bioavailability.

The oral bioavailability of commercial GLP-1 derivatives is very low.The oral bioavailability of GLP-1 derivatives under development for i.v.or s.c. administration is also low.

Accordingly, there is a need in the art for GLP-1 derivatives of animproved oral bioavailability. Such derivatives could be suitablecandidates for oral administration, as long as mainly their potency isgenerally satisfactory, and/or as long as their half-life is alsogenerally satisfactory.

Generally, the term bioavailability refers to the fraction of anadministered dose of an active pharmaceutical ingredient (API), such asa derivative of the invention that reaches the systemic circulationunchanged. By definition, when an API is administered intravenously, itsbioavailability is 100%. However, when it is administered via otherroutes (such as orally), its bioavailability decreases (due todegradation and/or incomplete absorption and first-pass metabolism).Knowledge about bioavailability is important when calculating dosagesfor non-intravenous routes of administration.

Absolute oral bioavailability compares the bioavailability (estimated asthe area under the curve, or AUC) of the API in systemic circulationfollowing oral administration, with the bioavailability of the same APIfollowing intravenous administration. It is the fraction of the APIabsorbed through non-intravenous administration compared with thecorresponding intravenous administration of the same API. The comparisonmust be dose normalised if different doses are used; consequently, eachAUC is corrected by dividing by the corresponding dose administered.

A plasma API concentration vs time plot is made after both oral andintravenous administration. The absolute bioavailability (F) is thedose-corrected AUC-oral divided by AUC-intravenous.

In a particular embodiment, the derivative of the invention has anabsolute oral bioavailability which is higher than that of semaglutide,preferably at least 10% higher, more preferably at least 20% higher,even more preferably at least 30% higher, or most preferably at least40% higher. In additional particular embodiments, it has an absoluteoral bioavailability which is at least 1.5 times that of semaglutide,preferably at least 2.0 times, more preferably at least 3.0 times, evenmore preferably at least 4.0 times, or most preferably at least 5.0times that of semaglutide.

Before testing oral bioavailability the derivatives of the invention maysuitably be formulated as is known in the art of oral formulations ofinsulinotropic compounds, e.g. using any one or more of the formulationsdescribed in WO 2008/145728.

In a particular embodiment, the derivatives of the invention have a verygood gastro intestinal stability. The gastro intestinal stability may bedetermined in vitro, by incubation of the derivative with a suitablydiluted extract from the gastro intestinal system from human beings, orfrom a relevant animal species such as minipig, LYD pig, dog, or rat,under physiologically relevant conditions, and for a physiologicallyrelevant period of time. The stability may be further improved when thederivative is combined with one or more relevant enzyme inhibitors, inan effective amount. Gastro intestinal stability in vitro may bemeasured using standard methods known in the art. The gastro intestinalstability of the derivatives of the invention is preferably improved ascompared to semaglutide, and/or as compared to the derivative of Example2 of WO 2011/080103 A1. The derivative of the invention is preferably atleast as stable as any one or both of these two comparative compounds,preferably the stability is improved by at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50%; more preferably by at least75%, at least 100%, at least 150%, at least 175%, or at least 200%—ascompared to the stability of any of these two comparative compounds.

Biophysical Properties

According to the fourth aspect, the derivatives of the invention havegood biophysical properties. These properties include but are notlimited to physical stability and/or solubility. These and otherbiophysical properties may be measured using standard methods known inthe art of protein chemistry. In a particular embodiment, theseproperties are improved as compared to native GLP-1 (SEQ ID NO: 1).Changed oligomeric properties of the derivatives may be at least partlyresponsible for the improved biophysical properties.

Additional particular embodiments of the derivatives of the inventionare described in the sections headed “PARTICULAR EMBODIMENTS” and“ADDITIONAL PARTICULAR EMBODIMENTS” before the experimental section.

Production Processes

The production of peptides like GLP-1 (7-37) and GLP-1 analogues is wellknown in the art.

The GLP-1 moiety of the derivatives of the invention, viz. K³⁷-GLP-1(7-37) or an analogue thereof, may for instance be produced by classicalpeptide synthesis, e.g., solid phase peptide synthesis using t-Boc orFmoc chemistry or other well established techniques, see, e.g., Greeneand Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons,1999, Florencio Zaragoza Dörwald, “Organic Synthesis on solid Phase”,Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis”,Edited by W. C. Chan and P. D. White, Oxford University Press, 2000.

Also, or alternatively, they may be produced by recombinant methods,viz. by culturing a host cell containing a DNA sequence encoding theanalogue and capable of expressing the peptide in a suitable nutrientmedium under conditions permitting the expression of the peptide.Non-limiting examples of host cells suitable for expression of thesepeptides are: Escherichia coli, Saccharomyces cerevisiae, as well asmammalian BHK or CHO cell lines.

Those derivatives of the invention which include non-natural amino acidsand/or a covalently attached N-terminal mono- or dipeptide mimetic maye.g. be produced as described in the experimental part. Or see e.g.,Hodgson et al: “The synthesis of peptides and proteins containingnon-natural amino acids”, Chemical Society Reviews, vol. 33, no. 7(2004), p. 422-430; and WO 2009/083549 A1 entitled “Semi-recombinantpreparation of GLP-1 analogues”.

Specific examples of methods of preparing a number of the derivatives ofthe invention are included in the experimental part.

Pharmaceutical Compositions

Pharmaceutical composition comprising a derivative of the invention or apharmaceutically acceptable salt, amide, or ester thereof, and apharmaceutically acceptable excipient may be prepared as is known in theart.

The term “excipient” broadly refers to any component other than theactive therapeutic ingredient(s). The excipient may be an inertsubstance, an inactive substance, and/or a not medicinally activesubstance.

The excipient may serve various purposes, e.g. as a carrier, vehicle,diluent, tablet aid, and/or to improve administration, and/or absorptionof the active substance.

The formulation of pharmaceutically active ingredients with variousexcipients is known in the art, see e.g. Remington: The Science andPractice of Pharmacy (e.g. 19^(th) edition (1995), and any latereditions).

Non-limiting examples of excipients are: Solvents, diluents, buffers,preservatives, tonicity regulating agents, chelating agents, andstabilisers.

Examples of formulations include liquid formulations, i.e. aqueousformulations, i.e. formulations comprising water. A liquid formulationmay be a solution, or a suspension. An aqueous formulation typicallycomprises at least 50% w/w water, or at least 60%, 70%, 80%, or even atleast 90% w/w of water.

Alternatively a pharmaceutical composition may be a solid formulation,e.g. a freeze-dried or spray-dried composition, which may be used as is,or whereto the physician or the patient adds solvents, and/or diluentsprior to use.

The pH in an aqueous formulation may be anything between pH 3 and pH 10,for example from about 7.0 to about 9.5; or from about 3.0 to about 7.0.

A pharmaceutical composition may comprise a buffer. The buffer may e.g.be selected from the group consisting of sodium acetate, sodiumcarbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine,sodium dihydrogen phosphate, disodium hydrogen phosphate, sodiumphosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malicacid, succinate, maleic acid, fumaric acid, tartaric acid, asparticacid, and mixtures thereof.

A pharmaceutical composition may comprise a preservative. Thepreservative may e.g. be selected from the group consisting of phenol,o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate,chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride,chlorphenesine (3p-chlorphenoxypropane-1,2-diol), and mixtures thereof.The preservative may be present in a concentration from 0.1 mg/ml to 20mg/ml.

A pharmaceutical composition may comprise an isotonic agent. Theisotonic agent may e.g. be selected from the group consisting of a salt(e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.glycine, histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), and mixtures thereof. Any sugar suchas mono-, di-, or polysaccharides, or water-soluble glucans, includingfor example fructose, glucose, mannose, sorbose, xylose, maltose,lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,alfa and beta HPCD, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. Sugar alcohol is defined as aC4-C8 hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol.

A pharmaceutical composition may comprise a chelating agent. Thechelating agent may e.g. be selected from salts ofethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid,and mixtures thereof.

A pharmaceutical composition may comprise a stabiliser. The stabilisermay e.g. be one or more oxidation inhibitors, aggregation inhibitors,surfactants, and/or one or more protease inhibitors.

The term “aggregate formation” refers to a physical interaction betweenthe polypeptide molecules resulting in formation of oligomers, which mayremain soluble, or large visible aggregates that precipitate from thesolution. Aggregate formation by a polypeptide during storage of aliquid pharmaceutical composition can adversely affect biologicalactivity of that polypeptide, resulting in loss of therapeutic efficacyof the pharmaceutical composition. Furthermore, aggregate formation maycause other problems such as blockage of tubing, membranes, or pumpswhen the polypeptide-containing pharmaceutical composition isadministered using an infusion system.

A pharmaceutical composition may comprise an amount of an amino acidbase sufficient to decrease aggregate formation of the peptide duringstorage of the composition. The term “amino acid base” refers to one ormore amino acids (such as methionine, histidine, imidazole, arginine,lysine, isoleucine, aspartic acid, tryptophan, threonine), or analoguesthereof. Any amino acid may be present either in its free base form orin its salt form. Any stereoisomer (i.e., L, D, or a mixture thereof) ofthe amino acid base may be present.

Methionine (or other sulphuric amino acids or amino acid analogous) maybe added to inhibit oxidation of methionine residues to methioninesulfoxide when the peptide is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. Any stereoisomer ofmethionine (L or D) or combinations thereof can be used.

A pharmaceutical composition may comprise a stabiliser selected from thegroup of high molecular weight polymers or low molecular compounds. Thestabiliser may e.g. be selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride).

A pharmaceutical composition may comprise additional stabilising agentssuch as, but not limited to, methionine and EDTA, which protect thepolypeptide against methionine oxidation, and a nonionic surfactant,which protects the polypeptide against aggregation associated withfreeze-thawing or mechanical shearing.

A pharmaceutical composition may comprise one or more surfactants, forexample a surfactant, at least one surfactant, or different surfactants.The term “surfactant” refers to any molecules or ions that are comprisedof a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic)part. The surfactant may e.g. be selected from the group consisting ofanionic surfactants, cationic surfactants, nonionic surfactants, and/orzwitterionic surfactants.

A pharmaceutical composition may comprise one or more proteaseinhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid),and/or benzamidine HCl.

Additional, optional, ingredients of a pharmaceutical compositioninclude, e.g., wetting agents, emulsifiers, antioxidants, bulkingagents, metal ions, oily vehicles, proteins (e.g., human serum albumin,gelatine), and/or a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine).

Still further, a pharmaceutical composition may be formulated as isknown in the art of oral formulations of insulinotropic compounds, e.g.using any one or more of the formulations described in WO 2008/145728.

An administered dose may contain from 0.01 mg-100 mg of the derivative,or from 0.01-50 mg, or from 0.01-20 mg, or from 0.01 mg-10 mg of thederivative.

The derivative may be administered in the form of a pharmaceuticalcomposition. It may be administered to a patient in need thereof atseveral sites, for example, at topical sites such as skin or mucosalsites; at sites which bypass absorption such as in an artery, in a vein,or in the heart; and at sites which involve absorption, such as in theskin, under the skin, in a muscle, or in the abdomen.

The route of administration may be, for example, lingual; sublingual;buccal; in the mouth; oral; in the stomach; in the intestine; nasal;pulmonary, such as through the bronchioles, the alveoli, or acombination thereof; parenteral, epidermal; dermal; transdermal;conjunctival; uretal; vaginal; rectal; and/or ocular. In a particularembodiment the route of administration is per oral.

A composition may be administered in several dosage forms, for exampleas a solution; a suspension; an emulsion; a microemulsion; multipleemulsions; a foam; a salve; a paste; a plaster; an ointment; a tablet; acoated tablet; a chewing gum; a rinse; a capsule such as hard or softgelatine capsules; a suppositorium; a rectal capsule; drops; a gel; aspray; a powder; an aerosol; an inhalant; eye drops; an ophthalmicointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; avaginal ointment; an injection solution; an in situ transformingsolution such as in situ gelling, setting, precipitating, and in situcrystallisation; an infusion solution; or as an implant. A compositionmay further be compounded in a drug carrier or drug delivery system,e.g. in order to improve stability, bioavailability, and/or solubility.A composition may be attached to such system through covalent,hydrophobic, and/or electrostatic interactions. The purpose of suchcompounding may be, e.g., to decrease adverse effects, achievechronotherapy, and/or increase patient compliance.

A composition may also be used in the formulation of controlled,sustained, protracting, retarded, and/or slow release drug deliverysystems.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal, or intravenous injection by means of asyringe, optionally a pen-like syringe, or by means of an infusion pump.

A composition may be administered nasally in the form of a solution, asuspension, or a powder; or it may be administered pulmonally in theform of a liquid or powder spray.

Transdermal administration is a still further option, e.g. byneedle-free injection, from a patch such as an iontophoretic patch, orvia a transmucosal route, e.g. buccally.

A composition may be a stabilised formulation. The term “stabilisedformulation” refers to a formulation with increased physical and/orchemical stability, preferably both. In general, a formulation must bestable during use and storage (in compliance with recommended use andstorage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

The treatment with a derivative according to the present invention mayalso be combined with one or more additional pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. Examples of these pharmacologically active substances are:Insulin, sulphonylureas, biguanides, meglitinides, glucosidaseinhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)inhibitors, inhibitors of hepatic enzymes involved in stimulation ofgluconeogenesis and/or glycogenolysis, glucose uptake modulators,compounds modifying the lipid metabolism such as antihyperlipidemicagents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides(GIP analogs), compounds lowering food intake, RXR agonists and agentsacting on the ATP-dependent potassium channel of the β-cells;Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide,repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, alatriopril, quinapril and ramipril, calcium channelblockers such as nifedipine, felodipine, nicardipine, isradipine,nimodipine, diltiazem and verapamil, and a-blockers such as doxazosin,urapidil, prazosin and terazosin; CART (cocaine amphetamine regulatedtranscript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, Y2receptor agonists, Y4 receptor agonits, mixed Y2/Y4 receptor agonists,MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosisfactor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP(corticotropin releasing factor binding protein) antagonists, urocortinagonists, β3 agonists, oxyntomodulin and analogues, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists, gastric inhibitory polypeptide agonists or antagonists,gastrin and gastrin analogs.

The treatment with a derivative according to this invention may also becombined with a surgery that influences the glucose levels, and/or lipidhomeostasis such as gastric banding or gastric bypass.

Pharmaceutical Indications

The present invention also relates to a derivative of the invention foruse as a medicament.

In particular embodiments, the derivative of the invention may be usedfor the following medical treatments, all preferably relating one way orthe other to diabetes:

(i) prevention and/or treatment of all forms of diabetes, such ashyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetesof the young), gestational diabetes, and/or for reduction of HbA1C;

(ii) delaying or preventing diabetic disease progression, such asprogression in type 2 diabetes, delaying the progression of impairedglucose tolerance (IGT) to insulin requiring type 2 diabetes, and/ordelaying the progression of non-insulin requiring type 2 diabetes toinsulin requiring type 2 diabetes;

(iii) improving β-cell function, such as decreasing β-cell apoptosis,increasing β-cell function and/or β-cell mass, and/or for restoringglucose sensitivity to β-cells;

(iv) prevention and/or treatment of cognitive disorders;

(v) prevention and/or treatment of eating disorders, such as obesity,e.g. by decreasing food intake, reducing body weight, suppressingappetite, inducing satiety; treating or preventing binge eatingdisorder, bulimia nervosa, and/or obesity induced by administration ofan antipsychotic or a steroid; reduction of gastric motility; and/ordelaying gastric emptying;

(vi) prevention and/or treatment of diabetic complications, such asneuropathy, including peripheral neuropathy; nephropathy; orretinopathy;

(vii) improving lipid parameters, such as prevention and/or treatment ofdyslipidemia, lowering total serum lipids; lowering HDL; lowering small,dense LDL; lowering VLDL: lowering triglycerides; lowering cholesterol;increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in ahuman; inhibiting generation of apolipoprotein a (apo(a)) in vitroand/or in vivo;

(iix) prevention and/or treatment of cardiovascular diseases, such assyndrome X; atherosclerosis; myocardial infarction; coronary heartdisease; stroke, cerebral ischemia; an early cardiac or earlycardiovascular disease, such as left ventricular hypertrophy; coronaryartery disease; essential hypertension; acute hypertensive emergency;cardiomyopathy; heart insufficiency; exercise tolerance; chronic heartfailure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis;mild chronic heart failure; angina pectoris; cardiac bypass reocclusion;intermittent claudication (atheroschlerosis oblitterens); diastolicdysfunction; and/or systolic dysfunction;

(ix) prevention and/or treatment of gastrointestinal diseases, such asinflammatory bowel syndrome; small bowel syndrome, or Crohn's disease;dyspepsia; and/or gastric ulcers;

(x) prevention and/or treatment of critical illness, such as treatmentof a critically ill patient, a critical illness poly-nephropathy (CIPNP)patient, and/or a potential CIPNP patient; prevention of criticalillness or development of CIPNP; prevention, treatment and/or cure ofsystemic inflammatory response syndrome (SIRS) in a patient; and/or forthe prevention or reduction of the likelihood of a patient sufferingfrom bacteraemia, septicaemia, and/or septic shock duringhospitalisation; and/or

(xi) prevention and/or treatment of polycystic ovary syndrome (PCOS).

In a particular embodiment, the indication is selected from the groupconsisting of (i)-(iii) and (v)-(iix), such as indications (i), (ii),and/or (iii); or indication (v), indication (vi), indication (vii),and/or indication (iix).

In another particular embodiment, the indication is (i). In a furtherparticular embodiment the indication is (v). In a still furtherparticular embodiment the indication is (iix).

The following indications are particularly preferred: Type 2 diabetes,and/or obesity.

PARTICULAR EMBODIMENTS

The following are particular embodiments of the invention:

1. A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 26 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), anda maximum of eight amino acid changes as compared to GLP-1 (7-37),

which derivative comprises two protracting moieties attached to saidfirst and second K residue, respectively, via a linker, wherein

the protracting moiety is selected from Chem. 2, and Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

HOOC—(CH₂)_(x)—CO—*,  Chem. 1:

in which x is an integer in the range of 8-16, and y is an integer inthe range of 6-13; and

the linker comprises

*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NR₁R₂]—CO—*,  Chem. 3a:

which is connected at its CO—* end to the epsilon amino group of thefirst or the second K residue of the GLP-1 analogue, and wherein q is aninteger in the range of 0-5, R₁ and R₂ independently represent *—H or*—CH₃, and w is an integer in the range of 0-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The derivative of embodiment 1, wherein w is 0.3. The derivative of any of embodiments 1-2, wherein the linkercomprises

*—NH—(CH₂)_(q)—CH(NR₁R₂)—CO—*.  Chem. 4a:

4. The derivative of any of embodiments 1-3, wherein q is an integer inthe range of 3-5.5. The derivative of any of embodiments 1-3, wherein q is 4.6. The derivative of any of embodiments 1-5, wherein R, and R₂ areidentical.7. The derivative of any of embodiments 1-6, wherein R, and R₂ bothrepresent *—H.8. The derivative of any of embodiments 1-7, wherein the linkercomprises Chem. 3: *—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NH₂]—CO—*.9. The derivative of any of embodiments 1-8, wherein the linkercomprises

*—NH—(CH₂)_(q)—CH(NH₂)—CO—*.  Chem. 4:

10. The derivative of any of embodiments 1-9, wherein Chem. 3a is adi-radical of lysine.11. The derivative of any of embodiments 1-10, wherein Chem. 4a is adi-radical of lysine.12. The derivative of any of embodiments 1-11, wherein Chem. 3 is adi-radical of lysine.13. The derivative of any of embodiments 1-12, wherein Chem. 4 is adi-radical of lysine.14. The derivative of any of embodiments 1-13, wherein the linkercomprises Chem. 6: *—NH—(CH₂)₄—CH(NH₂)—CO—*.15. The derivative of any of embodiments 1-14, wherein R₁ and R₂ bothrepresent *—CH₃.16. The derivative of any of embodiments 1-15, wherein Chem. 3a is adi-radical of an N^(α),N^(α)-dimethyl lysine residue(6-amino-(S)-2-(dimethylamino)hexanoyl).17. The derivative of any of embodiments 1-16, wherein Chem. 4a is adi-radical of an N^(α),N^(α)-dimethyl lysine residue.18. The derivative of any of embodiments 1-17, wherein the linkercomprises

*—NH—(CH₂)₄—CH(N(CH₃)₂)—CO—*.  Chem. 6a:

19. The derivative of any of embodiments 1-18, wherein the linkercomprises z times Chem. 3a, wherein z is an integer in the range of 1-2.20. The derivative of any of embodiments 1-19, wherein the linkercomprises z times Chem. 4a, wherein z is an integer in the range of 1-2.21. The derivative of any of embodiments 1-20, wherein the linkercomprises z times Chem. 3, wherein z is an integer in the range of 1-2.22. The derivative of any of embodiments 1-21, wherein the linkercomprises z times Chem. 4, wherein z is an integer in the range of 1-2.23. The derivative of any of embodiments 1-22, wherein the linkercomprises z times Chem. 6a, wherein z is an integer in the range of 1-2.24. The derivative of any of embodiments 1-23, wherein the linkercomprises z times Chem. 6, wherein z is an integer in the range of 1-2.25. The derivative of any of embodiments 1-24, wherein z is 1.26. The derivative of any of embodiments 1-25, wherein z is 2.27. The derivative of any of embodiments 1-26, wherein when z is 2 thetwo Chem. 3a, Chem. 4a, Chem. 3, Chem. 4, Chem. 6a, or Chem. 6 elementsare interconnected via an amide bond.28. The derivative of any of embodiments 1-27, wherein the linkercomprises

*—NH—(CH₂)₄—CH(NH₂)—CO—NH—(CH₂)₄—CH(NH₂)—CO—*.  2×Chem. 6:

29. The derivative of any of embodiments 1-28, wherein the linkercomprises

*—NH—(CH₂)₄—CH(N(CH₃))—CO—NH—(CH₂)₄—CH(N(CH₃)₂)—CO—*.  2×Chem. 6a:

30. The derivative of any of embodiments 1-29, wherein the Chem. 3a,Chem. 4a, Chem. 6, Chem. 6a, Chem. 3, Chem. 4, 2×Chem. 6, or 2×Chem. 6a,respectively, is a first linker element.31. The derivative of any of embodiments 1-30, wherein the linkercomprises a second linker element, Chem. 12:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.32. The derivative of embodiment 31, wherein k is 1.33. The derivative of any of embodiments 31-32, wherein n is 1.34. The derivative of any of embodiments 31-33, wherein the secondlinker element is Chem. 13:

35. The derivative of any of embodiments 31-34, wherein Chem. 13 isincluded m times, wherein m is 0, or an integer in the range of 1-2; orpreferably m is 0, 1, or 2.36. The derivative of any of embodiments 35-36, wherein m is 0.37. The derivative of any of embodiments 35-36, wherein m is 1.38. The derivative of any of embodiments 35-36, wherein m is 2.39. The derivative of any of embodiments 35-38, wherein, when m isdifferent from 1, the Chem. 13 elements are interconnected via amidebond(s).40. The derivative of any of embodiments 1-39, wherein the linkercomprises a third linker element of Chem. 14:

41. The derivative of embodiment 40, wherein Chem. 14 is included ptimes, wherein p is 0, or an integer in the range of 1-3.42. The derivative of embodiment 41, wherein p is 0.43. The derivative of embodiment 41, wherein p is 1.44. The derivative of embodiment 41, wherein p is 2.45. The derivative of embodiment 41, wherein p is 3.46. The derivative any of embodiments 40-45, wherein Chem. 14 is adi-radical of L-Glu.47. The derivative of any of embodiments 41-46, wherein, when p isdifferent from 0 and different from 1, the Chem. 14 elements areinterconnected via amide bond(s).48. The derivative of any of embodiments 1-47, wherein the linkercomprises a fourth linker element of Chem. 15:

*—NH—(CH₂)₇—CO—*.  Chem. 15:

49. The derivative of embodiment 48, wherein Chem. 15 is included stimes, wherein s is 0 or 1.50. The derivative of embodiment 49, wherein s is 0.51. The derivative of embodiment 49, wherein s is 1.52. The derivative of any of embodiments 1-51, wherein the linker andthe protracting moiety are interconnected via an amide bond.53. The derivative of any of embodiments 1-52, wherein the linker andthe GLP-1 analogue are interconnected via an amide bond.54. The derivative of any of embodiments 1-53, wherein the linker isattached to the epsilon-amino group of the first or the second Kresidue.55. The derivative of any of embodiments 1-54, wherein the linkerconsists of Chem. 14 and two times Chem. 6 (Chem.14-2×Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue.56. The derivative of any of embodiments 1-54, wherein the linkerconsists of two times Chem. 14 and two times Chem. 6(2×Chem.14-2×Chem.6), interconnected via amide bonds and in the sequenceindicated, connected at its *—NH end to the CO—* end of the protractingmoiety, and at its CO—* end to the epsilon amino group of the first orthe second K residue of the GLP-1 analogue.57. The derivative of any of embodiments 1-54, wherein the linkerconsists of three times Chem. 14 and Chem. 6 (3×Chem.14-Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue.58. The derivative of any of embodiments 1-54, wherein the linkerconsists of Chem. 14, two times Chem. 13, and Chem. 6(Chem.14-2×Chem.13-Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue.59. The derivative of any of embodiments 1-54, wherein the linkerconsists of two times Chem. 14, Chem. 13, and Chem. 6(2×Chem.14-Chem.13-Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue.60. The derivative of any of embodiments 1-54, wherein the linkerconsists of three times Chem. 14 and two times Chem. 6(3×Chem.14-2×Chem.6), interconnected via amide bonds and in the sequenceindicated, connected at its *—NH end to the CO—* end of the protractingmoiety, and at its CO—* end to the epsilon amino group of the first orthe second K residue of the GLP-1 analogue.61. The derivative of any of embodiments 1-54, wherein the linkerconsists of Chem. 15, Chem. 14, and two times Chem. 6 (Chem.15-Chem.14-2×Chem.6), interconnected via amide bonds and in the sequenceindicated, connected at its *—NH end to the CO—* end of the protractingmoiety, and at its CO—* end to the epsilon amino group of the first orthe second K residue of the GLP-1 analogue.62. The derivative of any of embodiments 1-54, wherein the linkerconsists of Chem. 14, two times Chem. 6a (Chem.14-2×Chem. 6a),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue.63. The derivative of any of embodiments 1-54, wherein the linkerconsists of(i) Chem. 14, two times Chem. 13, and two times Chem. 6(Chem.14-2×Chem.13-2×Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue;(ii) Chem. 14, Chem. 13, and two times Chem. 6(Chem.14-Chem.13-2×Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue; or(iii) two times Chem. 14, and Chem. 6 (2×Chem.14-Chem.6), interconnectedvia amide bonds and in the sequence indicated, connected at its *—NH endto the CO—* end of the protracting moiety, and at its CO—* end to theepsilon amino group of the first or the second K residue of the GLP-1analogue.64. The derivative of any of embodiments 1-63, wherein the protractingmoiety is Chem. 1.65. The derivative of embodiment 64, wherein x is an even number.66. The derivative of any of embodiments 64-65, wherein x is 12.67. The derivative of any of embodiments 64-65, wherein x is 14.68. The derivative of any of embodiments 64-65, wherein x is 12 or 14.69. The derivative of any of embodiments 64-65, wherein x is 16.70. The derivative of any of embodiments 1-69, wherein Chem. 1 isrepresented by Chem. 1a:

71. The derivative of any of embodiments 1-63, wherein the protractingmoiety is Chem. 2.72. The derivative of embodiment 71, wherein y is an odd number.73. The derivative of embodiment 71, wherein y is an even number.74. The derivative of any of embodiments 71-72, wherein y is 9.75. The derivative of any of embodiments 71-72, wherein y is 11.76. The derivative of any of embodiments 71 and 73, wherein y is 10.77. The derivative of any of embodiments 1-63 and 71-76, wherein Chem. 2is represented by

Chem. 2a:

78. The derivative of any of embodiments 1-77, wherein the twoprotracting moieties are substantially identical.79. The derivative of any of embodiments 1-78, wherein the twoprotracting moieties have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.80. The derivative of any of embodiments 1-79, wherein the two linkersare substantially identical.81. The derivative of any of embodiments 1-80, wherein the two linkershave a similarity of at least 0.5; preferably at least 0.6; morepreferably at least 0.7, or at least 0.8; even more preferably at least0.9; or most preferably at least 0.99, such as a similarity of 1.0.82. The derivative of any of embodiments 1-81, wherein the two sidechains consisting of protracting moiety and linker are substantiallyidentical.83. The derivative of any of embodiments 1-82, wherein the two sidechains consisting of protracting moiety and linker have a similarity ofat least 0.5; preferably at least 0.6; more preferably at least 0.7, orat least 0.8; even more preferably at least 0.9; or most preferably atleast 0.99, such as a similarity of 1.0.84. The derivative of any of embodiments 78-83, wherein the two chemicalstructures to be compared are represented as fingerprints, such as a)ECFP_6 fingerprints; b) UNITY fingerprints; and/or c) MDL fingerprints;and wherein for each of a), b) and c) the Tanimoto coefficient ispreferably used for calculating the similarity of the two fingerprints.85. The derivative of any of embodiments 1-84, wherein the first Kresidue is designated K²⁶.86. The derivative of any of embodiments 1-85, wherein the second Kresidue is designated K³⁷.87. The derivative of any of embodiments 1-86, wherein the positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.88. The derivative of any of embodiments 1-87, wherein the positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.89. The derivative of any of embodiments 1-88, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by handwriting and eyeballing.90. The derivative of any of embodiments 1-89, wherein the positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.91. The derivative of any of embodiments 1-90, wherein the positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.92. The derivative of any of embodiments 1-91, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by use of a standard protein or peptide alignment program.93. The derivative of any of embodiments 90-92, wherein the alignmentprogram is a Needleman-Wunsch alignment.94. The derivative of any of embodiments 90-93, wherein the defaultscoring matrix and the default identity matrix is used.95. The derivative of any of embodiments 90-94, wherein the scoringmatrix is BLOSUM62.96. The derivative of any of embodiments 90-95, wherein the penalty forthe first residue in a gap is −10 (minus ten).97. The derivative of any of embodiments 90-96, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).98. The derivative of any of embodiments 1-97, wherein the analoguecomprises no K residues other than the first and the second K residue.99. The derivative of any of embodiments 1-98, wherein 37K is includedamongst the maximum eight amino acid changes.100. The derivative of any of embodiments 1-99, which has a maximum ofseven amino acid changes as compared to GLP-1 (7-37), wherein theseseven change(s) is/are in addition to the change to a K residue at theposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1).101. The derivative of any of embodiments 1-100, wherein the amino acidchange(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1 (7-37) (SEQ ID NO: 1): Position 37, and,optionally, position 7, 8, 22, 30, 31, 34, and/or 38.102. The derivative of any of embodiments 1-101, wherein the amino acidchange(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1 (7-37) (SEQ ID NO: 1): Position 34 and 37,and, optionally, position 7, 8, 22, 30, 31, and/or 38.103. The derivative of any of embodiments 1-102, wherein the analoguecomprises K³⁷, and, optionally, at least one of the following additionalchanges: Imp⁷, Aib⁸, E²², E³⁰, H³¹, (H³⁴ or R³⁴ or Q³⁴), and/or E³⁸.104. The derivative of any of embodiments 1-103, wherein the analoguecomprises K³⁷, and (H³⁴, R³⁴, or 034).105. The derivative of any of embodiments 1-103, wherein the analoguecomprises K³⁷ and H³⁴.106. The derivative of any of embodiments 1-103, wherein the analoguecomprises K³⁷ and R³⁴.107. The derivative of any of embodiments 1-103, wherein the analoguecomprises K³⁷ and Q³⁴.108. The derivative of any of embodiments 1-81, wherein the analoguecomprises at least one of the following changes: Imp⁷, Aib⁸, E²², E³⁰,H³¹, and/or E³⁸.109. The derivative of any of embodiments 1-108, wherein the analoguecomprises Aib⁸.110. The derivative of any of embodiments 1-109, wherein the analoguecomprises E²².111. The derivative of any of embodiments 1-110, wherein the analoguecomprises H³¹.112. The derivative of any of embodiments 1-111, wherein the analoguecomprises Imp⁷.113. The derivative of any of embodiments 1-112, wherein the analoguecomprises E³⁰.114. The derivative of any of embodiments 1-113, wherein the analoguecomprises E³⁸.115. The derivative of any of embodiments 1-114, wherein the analoguecomprises E²² and E³⁰.116. The derivative of any of embodiments 1-115, wherein the analoguedoes not comprise Q³⁴.117. The derivative of any of embodiments 1-116, wherein the analoguedoes not comprise H³¹.118. The derivative of any of embodiments 1-117, wherein, fordetermination of the changes in the analogue, the amino acid sequence ofthe analogue is compared to the amino acid sequence of nativeGLP-1(7-37) (SEQ ID NO: 1).119. The derivative of any of embodiments 1-118, wherein, fordetermination of a position in an analogue which corresponds to aspecified position in native GLP-1(7-37) (SEQ ID NO: 1), the amino acidsequence of the analogue is compared to the amino acid sequence ofnative GLP-1(7-37) (SEQ ID NO: 1).120. The derivative of any of embodiments 1-119, wherein the comparisonof the amino acid sequence of the analogue with that of GLP-1(7-37) (SEQID NO: 1) is done by handwriting and eyeballing.121. The derivative of any of embodiments 1-120, wherein the comparisonof the amino acid sequence of the analogue with that of GLP-1(7-37) (SEQID NO: 1) is done by use of a standard protein or peptide alignmentprogram.122. The derivative of embodiment 121, wherein the alignment program isa Needleman-Wunsch alignment.123. The derivative of any of embodiments 121-122, wherein the defaultscoring matrix and the default identity matrix is used.124. The derivative of any of embodiments 121-123, wherein the scoringmatrix is BLOSUM62.125. The derivative of any of embodiments 121-124, wherein the penaltyfor the first residue in a gap is −10 (minus ten).126. The derivative of any of embodiments 121-125, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).127. The derivative of any of embodiments 121-126, wherein the positioncorresponding to any of the indicated positions of GLP-1(7-37) (SEQ IDNO: 1) is identified by handwriting and eyeballing.128. The derivative of any of embodiments 121-127, wherein the positioncorresponding to any of the indicated positions of GLP-1(7-37) (SEQ IDNO: 1) is identified as described for position 26 and position 37 in anyof embodiments 87-97.129. The derivative of any of embodiments 1-128, wherein the analoguehas a maximum of six amino acid changes.130. The derivative of any of embodiments 1-129, wherein the analoguehas a maximum of five amino acid changes.131. The derivative of any of embodiments 1-130, wherein the analoguehas a maximum of four amino acid changes.132. The derivative of any of embodiments 1-131, wherein the analoguehas a maximum of three amino acid changes.133. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of two amino acid changes.134. The derivative of any of embodiments 1-133, wherein the analoguehas a maximum of one amino acid change.135. The derivative of any of embodiments 1-134, wherein the analoguehas a minimum of one amino acid modification.136. The derivative of any of embodiments 1-135, wherein the analoguehas a minimum of two amino acid changes.137. The derivative of any of embodiments 1-136, wherein the analoguehas a minimum of three amino acid changes.138. The derivative of any of embodiments 1-137, wherein the analoguehas a minimum of four amino acid changes.139. The derivative of any of embodiments 1-138, wherein the analoguehas a minimum of five amino acid changes.140. The derivative of any of embodiments 1-139, wherein the analoguehas a minimum of six amino acid changes.141. The derivative of any of embodiments 1-140, wherein the analoguehas one amino acid change.142. The derivative of any of embodiments 1-141, wherein the analoguehas two amino acid changes.143. The derivative of any of embodiments 1-142, wherein the analoguehas three amino acid changes.144. The derivative of any of embodiments 1-143, wherein the analoguehas four amino acid changes.145. The derivative of any of embodiments 1-144, wherein the analoguehas five amino acid changes.146. The derivative of any of embodiments 1-145, wherein the analoguehas six amino acid changes.147. The derivative of any of embodiments 1-146, wherein the change(s)is/are, independently, substitution(s), addition(s), and/or deletion(s).148. The derivative of any of embodiments 1-109, wherein the change(s)is/are substitution(s), and/or addition(s).149. The derivative of any of embodiments 1-148, wherein the change(s)is/are substitution(s).150. The derivative of any of embodiments 1-148, wherein the change(s)is/are addition(s).151. The derivative of any of embodiments 1-110, wherein the analogue a)comprises a GLP-1 analogue of Formula I; and/or b) is a GLP-1 analogueof Formula I:

Formula I:Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Gu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Lys-Xaa₃₈,(SEQ ID NO: 11) wherein

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,a-fluoromethyl-histidine, a-methyl-histidine, 3-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Phe or Leu;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Val, or Leu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu or Met;

Xaa₂₂ is Gly, Glu, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₅ is Ala or Val;

Xaa₂₇ is Glu or Leu;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His;

Xaa₃₃ is Val;

Xaa₃₄ is Glu, Asn, Gly, Gin, Arg, or His;

Xaa₃₅ is Gly or Aib;

Xaa₃₆ is Arg or Gly; and

Xaa₃₈ is Ser, Gly, Ala, Glu, Pro, Arg, or absent.

152. The derivative of embodiment 151, wherein the peptide of Formula Iis an analogue of GLP-1(7-37) (SEQ ID NO: 1).153. The derivative of any of embodiments 151-152, wherein Xaa₇ is Hisor Imp; Xaa₈ is Aib; Xaa₁₂ is Phe; Xaa₁₆ is Val; Xaa₁₈ is Ser; Xaa₁₉ isTyr; Xaa₂₀ is Leu; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gin; Xaa₂₅ is Ala;Xaa₂₇ is Glu; Xaa₃₀ is Ala or Glu; Xaa₃₁ is Trp or His; Xaa₃₃ is Val;Xaa₃₄ is Gin, Arg, or His; Xaa₃₅ is Gly; Xaa₃₆ is Arg; and Xaa₃₈ is Gluor absent154. The derivative of any of embodiments 151-153, wherein Xaa₇ is His.155. The derivative of any of embodiments 151-153, wherein Xaa₇ is Imp.156. The derivative of any of embodiments 151-155, wherein Xaa₈ is Aib.157. The derivative of any of embodiments 151-156, wherein Xaa₁₂ is Phe.158. The derivative of any of embodiments 151-157, wherein Xaa₁₆ is Val.159. The derivative of any of embodiments 151-158, wherein Xaa₁₈ is Ser.160. The derivative of any of embodiments 151-159, wherein Xaa₁₉ is Tyr.161. The derivative of any of embodiments 151-160, wherein Xaa₂₀ is Leu.162. The derivative of any of embodiments 151-161, wherein Xaa₂₂ is Gly.163. The derivative of any of embodiments 151-161, wherein Xaa₂₂ is Glu.164. The derivative of any of embodiments 151-163, wherein Xaa₂₃ is Gin.165. The derivative of any of embodiments 151-164, wherein Xaa₂₅ is Ala.166. The derivative of any of embodiments 151-165, wherein Xaa₂₇ is Glu.167. The derivative of any of embodiments 151-166, wherein Xaa₃₀ is Ala.168. The derivative of any of embodiments 151-166, wherein Xaa₃₀ is Glu.169. The derivative of any of embodiments 151-168, wherein Xaa₃₀ is Glu.170. The derivative of any of embodiments 151-169, wherein Xaa₃₁ is Trp.171. The derivative of any of embodiments 151-170, wherein Xaa₃₁ is His.172. The derivative of any of embodiments 151-171, wherein Xaa₃₃ is Val.173. The derivative of any of embodiments 151-172, wherein Xaa₃₄ is Gin.174. The derivative of any of embodiments 151-172, wherein Xaa₃₄ is Arg.175. The derivative of any of embodiments 151-172, wherein Xaa₃₄ is His.176. The derivative of any of embodiments 151-175, wherein Xaa₃₅ is Gly.177. The derivative of any of embodiments 151-176, wherein Xaa₃₆ is Arg.178. The derivative of any of embodiments 151-177, wherein Xaa₃₈ isabsent.179. The derivative of any of embodiments 151-177, wherein Xaa₃₈ is Glu.180. The derivative of any of embodiments 1-179, wherein the analoguecomprises the following amino acid changes, as compared to GLP-1(7-37)(SEQ ID NO: 1):(i) 8Aib, 34R, 37K; (ii) 8Aib, 34H, 37K; (iii) 8Aib, 31H, 34Q, 37K; (iv)8Aib, 34Q, 37K(v) 8Aib, 22E, 34Q, 37K; (vi) 8Aib, 22E, 34R, 37K; (vii) 7Imp, 8Aib,34R, 37K; (iix) 8Aib, 22E, 30E, 34R, 37K, 38E; or (ix) 8Aib, 22E, 30E,34R, 37K.181. The derivative of any of embodiments 1-180, wherein the analoguehas the following amino acid changes, as compared to GLP-1(7-37) (SEQ IDNO: 1):(i) 8Aib, 34R, 37K; (ii) 8Aib, 34H, 37K; (iii) 8Aib, 31H, 34Q, 37K; (iv)8Aib, 34Q, 37K(v) 8Aib, 22E, 34Q, 37K; (vi) 8Aib, 22E, 34R, 37K; (vii) 7Imp, 8Aib,34R, 37K; (iix) 8Aib, 22E, 30E, 34R, 37K, 38E; or (ix) 8Aib, 22E, 30E,34R, 37K182. A compound, preferably according to any of embodiments 1-181, whichis(i) selected from the following:Chem. 21, Chem. 22, Chem. 23, Chem. 24, Chem. 25, Chem. 26, Chem. 27,Chem. 28, Chem. 29, Chem. 30, Chem. 31, Chem. 32, Chem. 33, Chem. 34,Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, Chem. 40, Chem. 41,Chem. 42, Chem. 43, Chem. 44, Chem. 45, Chem. 46, Chem. 47, Chem. 48,Chem. 49, Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55,Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, or Chem. 62;(ii) characterised by its name, and selected from a listing of each ofthe names of the compounds of Examples 1-43 herein; or(iii) a pharmaceutically acceptable salt, amide, or ester of thecompound of (i) and (ii); wherein preferably the compound of (ii) is acompound of (i).183. The derivative of any of embodiments 1-182, which has GLP-1activity.184. The derivative of embodiment 183, wherein GLP-1 activity refers tothe capability of activating the human GLP-1 receptor.185. The derivative of embodiment 184, wherein activation of the humanGLP-1 receptor is measured in an in vitro assay, as the potency of cAMPproduction.186. The derivative of any of embodiments 1-185, which has a potencycorresponding to an EC₅₀a) below 10000 pM, preferably below 8000 pM, more preferably below 5000pM, even more preferably below 4000 pM, or most preferably below 3000pM;b) below 2000 pM, preferably below 1200 pM, more preferably below 1000pM, even more preferably below 800 pM, or most preferably below 600 pM;c) below 400 pM, preferably below 300 pM, more preferably below 200 pM,even more preferably below 150 pM, or most preferably below 100 pM; ord) below 80 pM, preferably below 60 pM, more preferably below 50 pM,even more preferably below 40 pM, or most preferably below 30 pM.187. The derivative of embodiment 186, wherein the potency is determinedas EC₅₀ for stimulation of the formation of cAMP in a medium containingthe human GLP-1 receptor, such as a medium of the following composition(final in-assay concentrations) 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl₂,6H₂O; 150 mM NaCl; 0.01% Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uMGTP, preferably using a stable transfected cell-line such as BHK467-12A(tk-ts13), and/or using for the determination of cAMP a functionalreceptor assay, e.g. based on competition between endogenously formedcAMP and exogenously added biotin-labelled cAMP, in which assay cAMP ismore preferably captured using a specific antibody, and/or wherein aneven more preferred assay is the AlphaScreen cAMP Assay, most preferablythe one described in Example 44.188. The derivative of any of embodiments 183-187, wherein activation ofthe human GLP-1 receptor is measured in an in vitro assay, in a reportergene assay.189. The derivative of embodiment 188, wherein the assay is performed ina stably transfected BHK cell line that expresses the human GLP-1receptor and contains the DNA for the cAMP response element (CRE)coupled to a promoter and the gene for firefly luciferase (CREluciferase).190. The derivative of embodiment 189, wherein when assay incubation iscompleted luciferin is added and luminescence is measured.191. The derivative of any of embodiments 188-190, wherein the assay isperformed in the absence of serum albumin (0% HSA, final assayconcentration).192. The derivative of any of embodiments 188-191, wherein the assay isperformed in the presence of 1% serum albumin (HSA, final assayconcentration).193. The derivative of any of embodiments 188-192, wherein the cells areBHK cells with BHK-ts13 as a parent cell line.194. The derivative of any of embodiments 188-193, wherein the cells arederived from clone FCW467-12A.195. The derivative of any of embodiments 188-194, wherein the cells arecultured at 5% CO₂ in cell culture medium, aliquoted and stored inliquid nitrogen.196. The derivative of embodiment 195, wherein the cell culture mediumis 10% FBS (Fetal Bovine Serum), 1 mg/ml G418, 240 nM MTX (methotrexate)and 1% pen/strep (penicillin/streptomycin).197. The derivative of any of embodiments 188-196, wherein before eachassay a cell culture aliquot is taken up and washed twice in PBS beforebeing suspended at the desired concentration in assay buffer.198. The derivative of any of embodiments 188-197, wherein for 96-wellplates the suspension is made to give a final concentration of 5×10³cells/well.199. The derivative of any of embodiments 197-198, wherein the assaybuffer is 1% assay buffer, which consists of 2% ovalbumin, 0.2% PluronicF-68 and 2% HSA in assay medium.200. The derivative of any of embodiments 197-198, wherein the assaybuffer is 0% assay buffer, which consists of 2% ovalbumin and 0.2%Pluronic F-68 in assay medium.201. The derivative of any of embodiments 199-200, wherein assay mediumconsists of DMEM w/o phenol red, 10 mM Hepes and 1× Glutamax.202. The derivative of any of embodiments 188-201, wherein the assayprocedure comprises the following steps:i) Cell stocks are thawed in a 37° C. water bath;ii) cells are washed three times in PBS;iii) the cells are counted and adjusted to 5×10³ cells/50 μl (1×10⁵cells/ml) in assay medium, and a 50 μl aliquot of cells is transferredto each well in the assay plate;iv) stocks of the test compounds and reference compounds, if any, arediluted to a concentration of 0.2 pM in either 0% assay buffer for the0% HSA assay or 1% assay buffer for the 1% HSA assay; and compounds arediluted 10-fold to give a suitable range of concentrations (such as:2×10⁻⁷ M, 2×10⁻⁸ M; 2×10⁻⁹ M, 2×10⁻¹⁰ M, 2×10⁻¹¹ M, 2×10⁻¹² M and2×10⁻¹³ M), and for each compound a blank assay buffer control is alsoincluded;v) a 50 μl aliquot of compound or blank is transferred in triplicatefrom the dilution plate to the assay plate, and compounds are tested atsuitable concentrations (such as the following final concentrations:1×10⁻⁷ M, 1×10⁻⁸ M; 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M and1×10⁻¹³ M);vi) the assay plate is incubated for 3 h in a 5% CO₂ incubator at 37°C.;vii) the assay plate is removed from the incubator and allowed to standat room temperature for 15 min;ixx) a 100 μl aliquot of luciferin (such as steadylite plus reagent) isadded to each well of the assay plate;ix) each assay plate is covered to protect it from light and shaken for30 min at room temperature; andx) each assay plate is read, for example in a Packard TopCount NXTinstrument.203. The derivative of embodiment 202, wherein the data from theTopCount instrument are transferred to GraphPad Prism 5 software fordesired calculations.204. The derivative of any of embodiments 188-203, wherein values foreach triplicate is averaged, a non-linear regression performed, and theEC₅₀ values calculated.205. The derivative of any of embodiments 202-204, wherein theregression is (log(agonist) vs response-Variable slope (fourparameter)).206. The derivative of any of embodiments 204-205, wherein the potencyis determined as described in any of embodiments 188-205.207. The derivative of embodiment 206, wherein the potency is determinedas described in Example 45.208. The derivative of any of embodiments 183-207, which has a potencycorresponding to an EC₅₀ at 0% HSA ofa) below 400 pM, preferably below 300 pM, more preferably below 200 pM,even more preferably below 150 pM, or most preferably below 100 pM;b) below 80 pM, preferably below 60 pM, more preferably below 50 pM,even more preferably below 40 pM, or most preferably below 30 pM;c) below 25 pM, preferably below 20 pM, more preferably below 15 pM,even more preferably below 10 pM, or most preferably below 8.0 pM; ord) below 6.0 pM, preferably below 5.0 pM, more preferably below 4.0 pM,even more preferably below 3.0 pM, or most preferably below 2.0 pM.209. The derivative of any of embodiments 188-208, the EC₅₀ value ofwhich is no more than 20 times the EC₅₀ value for semaglutide.210. The derivative of any of embodiments 188-209, the EC₅₀ value ofwhich is no more than 15 times the EC₅₀ value for semaglutide.211. The derivative of any of embodiments 188-210, the EC₅₀ value ofwhich is no more than 10 times the EC₅₀ value for semaglutide.212. The derivative of any of embodiments 188-211, the EC₅₀ value ofwhich is no more than 5 times the EC₅₀ value for semaglutide.213. The derivative of any of embodiments 188-212, the EC₅₀ value ofwhich is no more than 2.5 times the EC₅₀ value for semaglutide.214. The derivative of any of embodiments 188-213, the EC₅₀ value ofwhich is lower than the EC₅₀ value for semaglutide.215. The derivative of any of embodiments 188-214, the EC₅₀ value ofwhich is less than 0.75 times the EC₅₀ value for semaglutide.216. The derivative of any of embodiments 188-215, the EC₅₀ value ofwhich is less than 0.50 times the EC₅₀ value for semaglutide.217. The derivative of any of embodiments 188-216, the EC₅₀ value ofwhich is less than 0.60 times the EC₅₀ value for a comparator compoundwhich is identical to the derivative in question except that the linkerconsists of Chem. 14 and two times Chem. 13, interconnected via amidebonds and in the sequence indicated, connected at its *—NH end to theCO—* end of the protracting moiety, and at its CO—* end to the epsilonamino group of the first or the second K residue of the GLP-1 analogue;wherein the two EC₅₀ values are determined in the same in vitro potencyassay, preferably as described in any of embodiments 185-187.218. The derivative of embodiment 217, wherein the EC₅₀ value of thederivative is less than 0.50 times the EC₅₀ value for the comparatorcompound.219. The derivative of embodiment 217, wherein the EC₅₀ value of thederivative is less than 0.40 times the EC₅₀ value for the comparatorcompound.220. The derivative of embodiment 217, wherein the EC₅₀ value of thederivative is less than 0.30 times the EC₅₀ value for the comparatorcompound.221. The derivative of embodiment 217, wherein the EC₅₀ value of thederivative is less than 0.20 times the EC₅₀ value for the comparatorcompound.222. The derivative of any of embodiments 188-221, the EC₅₀ value ofwhich is less than or equal to 0.61 times the EC₅₀ value for acomparator compound which is identical to the derivative in questionexcept that the linker consists of Chem. 14 and two times Chem. 13,interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue; wherein the two EC₅₀ values are determined in thesame in vitro potency assay, preferably as described in any ofembodiments 188-205, and in the absence of human serum albumin in theassay (0% HSA, final assay concentration).223. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.60 times the EC₅₀ value for the comparatorcompound.224. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.50 times the EC₅₀ value for the comparatorcompound.225. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.40 times the EC₅₀ value for the comparatorcompound.226. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.30 times the EC₅₀ value for the comparatorcompound.227. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.20 times the EC₅₀ value for the comparatorcompound.228. The derivative of embodiment 222, wherein the EC₅₀ value of thederivative is less than 0.10 times the EC₅₀ value for the comparatorcompound.229. The derivative of any of embodiments 1-228, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 0.001% HSA (lowalbumin) isa) below 500 nM, preferably below 250 nM, more preferably below 100 nM,or most preferably below 50 nM;b) below 10 nM, preferably below 8.0 nM, still more preferably below 6.0nM, even more preferably below 5.0 nM, or most preferably below 2.00 nM;c) below 1.00 nM, preferably below 0.80 nM, even more preferably below0.60 nM, or most preferably below 0.50 nM; ord) below 0.40 nM, preferably below 0.30 nM, even more preferably below0.20 nM, or most preferably below 0.10 nM.230. The derivative of embodiment 229, wherein activation of the humanGLP-1 receptor is measured as GLP-1 receptor binding affinity (IC₅₀) inthe presence of 0.001% HSA (low albumin).231. The derivative of any of embodiments 1-230, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 2.0% HSA (highalbumin) isa) below 1000 nM, preferably below 800 nM;b) below 700 nM, preferably below 500 nM, more preferably below 300 nM;orc) below 200 nM, preferably below 100 nM, more preferably below 50 nM,even more preferably below 40 nM, still more preferably below 30 nM, ormost preferably below 20 nM.232. The derivative of any of embodiments 229-231, wherein the bindingaffinity to the GLP-1 receptor is measured by way of displacement of¹²⁵I-GLP-1 from the receptor, preferably using a SPA binding assay.233. The derivative of embodiment 232, wherein the GLP-1 receptor isprepared using a stable, transfected cell line, preferably a hamstercell line, more preferably a baby hamster kidney cell line, such as BHKtk-ts13.234. The derivative of any of embodiments 229-233, wherein the IC₅₀value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.235. The derivative of any of embodiments 229-234, wherein the GLP-1receptor binding affinity (IC₅₀) is determined as described in Example46.236. The derivative of any of embodiments 229-235, the IC₅₀ value ofwhich is less than 0.50 times the IC₅₀ value for a comparator compoundwhich is identical to the derivative in question except that the linkerconsists of Chem. 14 and two times Chem. 13 (gGlu-2×OEG), interconnectedvia amide bonds and in the sequence indicated, connected at its *—NH endto the CO—* end of the protracting moiety, and at its CO—* end to theepsilon amino group of the first or the second K residue of the GLP-1analogue; wherein the two IC₅₀ values are determined in the same GLP-1receptor binding assay, preferably as described in any of embodiments229-230 and 232-235, and with a low concentration of human serum albuminin the assay (0.001% HSA, final assay concentration).237. The derivative of embodiment 236, wherein the IC₅₀ value of thederivative is less than 0.40 times the IC₅₀ value for the comparatorcompound.238. The derivative of embodiment 236, wherein the IC₅₀ value of thederivative is less than 0.30 times the IC₅₀ value for the comparatorcompound.239. The derivative of embodiment 236, wherein the IC₅₀ value of thederivative is less than 0.20 times the EC₅₀ value for the comparatorcompound.240. The derivative of embodiment 236, wherein the IC₅₀ value of thederivative is less than 0.10 times the EC₅₀ value for the comparatorcompound.241. The derivative of any of embodiments 1-240, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of semaglutide.242. The derivative of any of embodiments 1-241, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of liraglutide.243. The derivative of any of embodiments 1-242, wherein the derivativeis effective at lowering blood glucose in vivo in db/db mice.244. The derivative of any of embodiments 1-243, wherein the derivativeis effective at lowering body weight in vivo in db/db mice.245. The derivative of any of embodiments 1-244 which, in a PD study inpigs, reduces food intake on day 1, 2, 3, and/or 4 after s.c.administration of a single dose of the derivative, as compared to avehicle-treated control group, wherein preferably the dose is 0.3, 1, 3,10 or 30 nmol/kg; more preferably 3.0 nmol/kg.246. The derivative of embodiment 245, wherein the food intake on day 1is 80% or lower, preferably 60% or lower, more preferably 50% or lower,even more preferably 40% or lower, still more preferably 30% or lower,or most preferably 15% or lower; wherein the percentage is relative tothe food intake of the control group.247. The derivative of any of embodiments 245-246, wherein the foodintake on day 2 is 80% or lower, preferably 60% or lower, morepreferably 40% or lower, or most preferably 20% or lower, wherein thepercentage is relative to the food intake of the control group.248. The derivative of any of embodiments 245-247, wherein the foodintake on day 3 is 90% or lower, preferably 80% or lower, or mostpreferably 75% or lower, wherein the percentage is relative to the foodintake of the control group.249. The derivative of any of embodiments 245-248, wherein the study isconducted and the data compiled and analysed as described in Example 49.250. The derivative of any of embodiments 1-249, which has a moreprotracted profile of action than liraglutide.251. The derivative of embodiment 250, wherein protraction meanshalf-life in vivo in a relevant animal species, such as db/db mice, rat,pig, and/or, preferably, minipig; wherein the derivative is administeredi) s.c., and/or, ii) i.v.; preferably ii) i.v.252. The derivative of any of embodiments 1-251, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is higher than thatof semaglutide.253. The derivative of any of embodiments 1-252, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least a) 25%higher, b) 50% higher, c) 75% higher, d) 100% higher (=twice) ), or e)150% higher than the terminal half-life of semaglutide.254. The derivative of any of embodiments 1-253, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least threetimes the terminal half-life of semaglutide.255. The derivative of any of embodiments 1-254, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least fourtimes the terminal half-life of semaglutide.256. The derivative of any of embodiments 1-255, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least fivetimes the terminal half-life of semaglutide.257. The derivative of any of embodiments 250-256, wherein the half-lifeis determined in in vivo pharmacokinetic studies in rat, for example asdescribed in Example 48.258. The derivative of any of embodiments 1-257, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in minipigs isa) at least 8 hours, preferably at least 16 hours, more preferably atleast 24 hours, even more preferably at least 32 hours, or mostpreferably at least 40 hours;b) at least 50 hours, preferably at least 60 hours, more preferably atleast 70 hours, even more preferably at least 80 hours, or mostpreferably at least 90 hours; orc) at least 95 hours, preferably at least 100 hours, even morepreferably at least 105 hours, or most preferably at least 115 hours.259. The derivative of embodiment 258, wherein the minipigs are maleGöttingen minipigs.260. The derivative of any of embodiments 258-259, wherein the minipigsare 7-14 months of age, and preferably weighing from 16-35 kg.261. The derivative of any of embodiments 258-260, wherein the minipigsare housed individually, and fed once or twice daily, preferably withSDS minipig diet.262. The derivative of any of embodiments 258-261, wherein thederivative is dosed, i.v., after at least 2 weeks of acclimatisation.263. The derivative of any of embodiments 258-262, wherein the animalsare fasted for approximately 18 h before dosing and from 0 to 4 h afterdosing, and have ad libitum access to water during the whole period.264. The derivative of any of embodiments 258-263, wherein the GLP-1derivative is dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a suitable concentration, preferablyfrom 20-60 nmol/ml.265. The derivative of any of embodiments 258-264, wherein intravenousinjections of the derivative are given in a volume corresponding to 1-2nmol/kg.266. The derivative of any of embodiments 258-265, wherein the terminalhalf-life (T_(1/2)) is determined in in vivo pharmacokinetic studies inminipig, for example as described in Example 47.267. An intermediate product in the form of a GLP-1 analogue whichcomprises the following changes as compared to GLP-1(7-37) (SEQ ID NO:1): (ii) 8Aib, 34H, 37K; (v) 8Aib, 22E, 34Q, 37K; (vii) 7Imp, 8Aib, 34R,37K; (iix) 8Aib, 22E, 30E, 34R, 37K, 38E; or (ix) 8Aib, 22E, 30E, 34R,37K; or a pharmaceutically acceptable salt, amide, or ester of any ofthe analogues of (ii), (v), (vii), (iix), or (ix).268. An intermediate product in the form of a GLP-1 analogue selectedfrom the following analogues of GLP-1(7-37) (SEQ ID NO: 1): (ii) 8Aib,34H, 37K; (v) 8Aib, 22E, 34Q, 37K; (vi) 8Aib, 22E, 34R, 37K; (vii) 7Imp,8Aib, 34R, 37K; (iix) 8Aib, 22E, 30E, 34R, 37K, 38E; and (ix) 8Aib, 22E,30E, 34R, 37K; or a pharmaceutically acceptable salt, amide, or ester ofany of the analogues of (ii), (v), (vi), (vii), (iix), or (ix).269. The analogue of any of embodiments 267-268, wherein the comparisonwith GLP-1(7-37) (SEQ ID NO: 1) is made by handwriting and eyeballing.270. The analogue of any of embodiments 267-269, wherein the comparisonwith GLP-1(7-37) (SEQ ID NO: 1) is made by use of a standard protein orpeptide alignment program.271. The analogue of embodiment 270, wherein the alignment program is aNeedleman-Wunsch alignment.272. The analogue of any of embodiments 270-271, wherein the defaultscoring matrix and the default identity matrix is used.273. The analogue of any of embodiments 270-272, wherein the scoringmatrix is BLOSUM62.274. The analogue of any of embodiments 270-273, wherein the penalty forthe first residue in a gap is −10 (minus ten).275. The analogue of any of embodiments 270-274, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).276. A compound selected from(S)-2-Dimethylamino-6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoicacid; and

Chem. 63:

or a pharmaceutically acceptable salt, amide, or ester thereof.277. A derivative according to any of embodiments 1-266, or an analogueaccording to any of embodiments 267-275, for use as a medicament.278. A derivative according to any of embodiments 1-266, or an analogueaccording to any of embodiments 267-275, for use in the treatment and/orprevention of all forms of diabetes and related diseases, such as eatingdisorders, cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.279. Use of a derivative according to any of embodiments 1-266, or ananalogue according to any of embodiments 267-275, in the manufacture ofa medicament for the treatment and/or prevention of all forms ofdiabetes and related diseases, such as eating disorders, cardiovasculardiseases, gastrointestinal diseases, diabetic complications, criticalillness, and/or polycystic ovary syndrome; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression.280. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of embodiments 1-266, or an analogueaccording to any of embodiments 267-275.

The invention also relates to a derivative of a GLP-1 analogue, whichanalogue comprises a first K residue at a position corresponding toposition 26 of GLP-1 (7-37) (SEQ ID NO: 1), a second K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), anda maximum of eight amino acid changes as compared to GLP-1(7-37), whichderivative comprises a first and a second protracting moiety attached tosaid first and second K residue, respectively, via a first and a secondlinker, respectively, wherein the first and the second protractingmoiety is selected from Chem. 2, and Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

HOOC—(CH₂)_(x)—CO—*,  Chem. 1:

in which x is an integer in the range of 8-16, and y is an integer inthe range of 6-13; and the first and the second linker comprises Chem.3a:

*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NR₁R₂]—CO—*,  Chem. 3a:

which is connected at its CO—* end to the epsilon amino group of thefirst or the second K residue of the GLP-1 analogue, and wherein q is aninteger in the range of 0-5, R₁ and R₂ independently represent *—H (ahydrogen radical) or *—CH₃ (methyl), and w is an integer in the range of0-5; or a pharmaceutically acceptable salt, amide, or ester thereof;

as well as any of the above embodiments 2-280 appended hereto asdependent embodiments.

ADDITIONAL PARTICULAR EMBODIMENTS

1. A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 26 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), anda maximum of eight amino acid changes as compared to GLP-1(7-37),

which derivative comprises two protracting moieties attached to saidfirst and second K residue, respectively, via a linker, wherein

the protracting moiety is selected from Chem. 2, and Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

HOOC—(CH₂)_(x)—CO—*,  Chem. 1:

in which x is an integer in the range of 8-14, and y is an integer inthe range of 6-13; and

the linker comprises

*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NH₂]—CO—*,  Chem. 3:

which is connected at its CO—* end to the epsilon amino group of thefirst or the second K residue of the GLP-1 analogue, and wherein q is aninteger in the range of 0-5, and w is an integer in the range of 0-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The derivative of embodiment 1, wherein the linker comprises z timesChem. 3, wherein z is an integer in the range of 1-2.3. The derivative of embodiment 2, wherein z is 1.4. The derivative of embodiment 2, wherein z is 2.5. The derivative of any of embodiments 2 and 4, wherein when z is 2 theChem. 3 elements are interconnected via an amide bond.6. The derivative of any of embodiments 1-5, wherein w is 0.7. The derivative of any of embodiments 1-6, wherein q is 4.8. The derivative of any of embodiments 1-7, wherein the linkercomprises

*—NH—(CH₂)_(q)—CH(NH₂)—CO—*,  Chem. 4:

wherein q is an integer in the range of β-5.9. The derivative of any of embodiments 1-8, wherein q is 4.10. The derivative of any of embodiments 1-9, wherein Chem. 3, or Chem.4, respectively, is a di-radical of lysine.11. The derivative of any of embodiments 1-10, wherein the linkercomprises

*—NH—(CH₂)₄—CH(NH₂)—CO—*.  Chem. 6:

12. The derivative of any of embodiments 1-11, wherein the linkercomprises 2×Chem. 6: *—NH—(CH₂)₄—CH(NH₂)—CO—NH—(CH₂)₄—CH(NH₂)—CO—*.13. The derivative of any of embodiments 1-12, wherein Chem. 3, Chem. 4,Chem. 6, or 2×Chem. 6, respectively, is a first linker element.14. The derivative of any of embodiments 1-13, wherein the linkercomprises a second linker element, Chem. 12:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.15. The derivative of embodiment 14, wherein k is 1.16. The derivative of any of embodiments 14-15, wherein n is 1.17. The derivative of any of embodiments 14-16, wherein the secondlinker element is Chem. 13:

18. The derivative of any of embodiments 14-17, wherein Chem. 13 isincluded m times, wherein m is 0, or an integer in the range of 1-2.19. The derivative of embodiment 18, wherein m is 0, 1, or 2.20. The derivative of any of embodiments 18-19, wherein m is 0.21. The derivative of any of embodiments 18-19, wherein m is 1.22. The derivative of any of embodiments 18-19, wherein m is 2.23. The derivative of any of embodiments 18-22, wherein, when m isdifferent from 1, the Chem. 13 elements are interconnected via amidebond(s).24. The derivative of any of embodiments 1-23, wherein the linkercomprises a third linker element of Chem. 14:

25. The derivative of embodiment 24, wherein Chem. 14 is included ptimes, wherein p is 0, or an integer in the range of 1-3.26. The derivative of embodiment 25, wherein p is 0.27. The derivative of embodiment 25, wherein p is 1.28. The derivative of embodiment 25, wherein p is 2.29. The derivative of embodiment 25, wherein p is 3.30. The derivative any of embodiments 24-29, wherein Chem. 14 is adi-radical of L-Glu.31. The derivative of any of embodiments 25-30, wherein, when p isdifferent from 0 and different from 1, the Chem. 14 elements areinterconnected via amide bond(s).32. The derivative of any of embodiments 1-31, wherein the linker andthe protracting moiety are interconnected via an amide bond.33. The derivative of any of embodiments 1-32, wherein the linker andthe GLP-1 analogue are interconnected via an amide bond.34. The derivative of any of embodiments 1-33, wherein the linker isattached to the epsilon-amino group of the first or the second Kresidue.35. The derivative of any of embodiments 1-34, wherein the linkerconsists of Chem. 14 and two times Chem. 6 (Chem.14-2×Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue.36. The derivative of any of embodiments 1-34, wherein the linkerconsists of two times Chem. 14 and two times Chem. 6(2×Chem.14-2×Chem.6), interconnected via amide bonds and in the sequenceindicated, connected at its *—NH end to the CO—* end of the protractingmoiety, and at its CO—* end to the epsilon amino group of the first orthe second K residue of the GLP-1 analogue.37. The derivative of any of embodiments 1-34, wherein the linkerconsists of three times Chem. 14 and Chem. 6 (3×Chem.14-Chem.6),interconnected via amide bonds and in the sequence indicated, connectedat its *—NH end to the CO—* end of the protracting moiety, and at itsCO—* end to the epsilon amino group of the first or the second K residueof the GLP-1 analogue.38. The derivative of any of embodiments 1-34, wherein the linkerconsists of Chem. 14, two times Chem. 13, and Chem. 6(Chem.14-2×Chem.13-Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue.39. The derivative of any of embodiments 1-34, wherein the linkerconsists of two times Chem. 14, Chem. 13, and Chem. 6(2×Chem.14-Chem.13-Chem.6), interconnected via amide bonds and in thesequence indicated, connected at its *—NH end to the CO—* end of theprotracting moiety, and at its CO—* end to the epsilon amino group ofthe first or the second K residue of the GLP-1 analogue.40. The derivative of any of embodiments 1-39, wherein the protractingmoiety is Chem. 1.41. The derivative of embodiment 40, wherein x is an even number.42. The derivative of any of embodiments 40-41, wherein x is 12.43. The derivative of any of embodiments 40-41, wherein x is 14.44. The derivative of any of embodiments 1-39, wherein the protractingmoiety is Chem. 2.45. The derivative of embodiment 44, wherein y is an odd number.46. The derivative of embodiment 44, wherein y is an even number.47. The derivative of any of embodiments 44-45, wherein y is 9.48. The derivative of any of embodiments 44-45, wherein y is 11.49. The derivative of any of embodiments 44 and 46, wherein y is 10.50. The derivative of any of embodiments 1-43, wherein Chem. 1 isrepresented by Chem. 1a:

51. The derivative of any of embodiments 1-39 and 44-49, wherein Chem. 2is represented by

Chem. 2a:

52. The derivative of any of embodiments 1-51, wherein the twoprotracting moieties are substantially identical.53. The derivative of any of embodiments 1-52, wherein the twoprotracting moieties have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.54. The derivative of any of embodiments 1-53, wherein the two linkersare substantially identical.55. The derivative of any of embodiments 1-54, wherein the two linkershave a similarity of at least 0.5; preferably at least 0.6; morepreferably at least 0.7, or at least 0.8; even more preferably at least0.9; or most preferably at least 0.99, such as a similarity of 1.0.56. The derivative of any of embodiments 1-55, wherein the two sidechains consisting of protracting moiety and linker are substantiallyidentical.57. The derivative of any of embodiments 1-56, wherein the two sidechains consisting of protracting moiety and linker have a similarity ofat least 0.5; preferably at least 0.6; more preferably at least 0.7, orat least 0.8; even more preferably at least 0.9; or most preferably atleast 0.99, such as a similarity of 1.0.58. The derivative of any of embodiments 52-57, wherein the two chemicalstructures to be compared are represented as fingerprints, such as a)ECFP_6 fingerprints; b) UNITY fingerprints; and/or c) MDL fingerprints;and wherein for each of a), b) and c) the Tanimoto coefficient ispreferably used for calculating the similarity of the two fingerprints.59. The derivative of any of embodiments 1-58, wherein the first Kresidue is designated K²⁶.60. The derivative of any of embodiments 1-59, wherein the second Kresidue is designated K³⁷.61. The derivative of any of embodiments 1-60, wherein the positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.62. The derivative of any of embodiments 1-61, wherein the positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.63. The derivative of any of embodiments 1-62, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by handwriting and eyeballing.64. The derivative of any of embodiments 1-63, wherein the positioncorresponding to position 26 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.65. The derivative of any of embodiments 1-64, wherein the positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.66. The derivative of any of embodiments 1-65, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by use of a standard protein or peptide alignment program.67. The derivative of any of embodiments 64-66, wherein the alignmentprogram is a Needleman-Wunsch alignment.68. The derivative of any of embodiments 64-67, wherein the defaultscoring matrix and the default identity matrix is used.69. The derivative of any of embodiments 64-68, wherein the scoringmatrix is BLOSUM62.70. The derivative of any of embodiments 64-69, wherein the penalty forthe first residue in a gap is −10 (minus ten).71. The derivative of any of embodiments 64-70, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).72. The derivative of any of embodiments 1-71, wherein the analoguecomprises no K residues other than the first and the second K residue.73. The derivative of any of embodiments 1-72, wherein 37K is includedamongst the maximum eight amino acid changes.74. The derivative of any of embodiments 1-72, which has a maximum ofseven amino acid changes as compared to GLP-1 (7-37), wherein theseseven change(s) is/are in addition to the change to a K residue at theposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1).75. The derivative of any of embodiments 1-74, wherein the amino acidchange(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1 (7-37) (SEQ ID NO: 1): Position 37, and,optionally, position 8, 22, 31, and/or 34.76. The derivative of any of embodiments 1-75, wherein the amino acidchange(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1 (7-37) (SEQ ID NO: 1): Position 34 and 37,and, optionally, position 8, 22, and/or 31.77. The derivative of any of embodiments 1-76, wherein the analoguecomprises K³⁷, and, optionally, at least one of the following additionalchanges: Aib⁸, E²², H³¹, and/or (H³⁴ or R³⁴ or Q³⁴).78. The derivative of any of embodiments 1-77, wherein the analoguecomprises K³⁷, and (H³⁴, R³⁴, or Q³⁴).79. The derivative of any of embodiments 1-78, wherein the analoguecomprises K³⁷ and H³⁴.80. The derivative of any of embodiments 1-78, wherein the analoguecomprises K³⁷ and R³⁴.81. The derivative of any of embodiments 1-78, wherein the analoguecomprises K³⁷ and Q³⁴.82. The derivative of any of embodiments 1-81, wherein the analoguecomprises at least one of the following changes: Aib⁸, E²², and/or H³¹.83. The derivative of any of embodiments 1-82, wherein the analoguecomprises Aib⁸.84. The derivative of any of embodiments 1-83, wherein the analoguecomprises E²².85. The derivative of any of embodiments 1-84, wherein the analoguecomprises H³¹.86. The derivative of any of embodiments 1-85, wherein, fordetermination of the changes in the analogue, the amino acid sequence ofthe analogue is compared to the amino acid sequence of nativeGLP-1(7-37) (SEQ ID NO: 1).87. The derivative of any of embodiments 1-86, wherein, fordetermination of a position in an analogue which corresponds to aspecified position in native GLP-1(7-37) (SEQ ID NO: 1), the amino acidsequence of the analogue is compared to the amino acid sequence ofnative GLP-1(7-37) (SEQ ID NO: 1).88. The derivative of any of embodiments 1-87, wherein the comparison ofthe amino acid sequence of the analogue with that of GLP-1(7-37) (SEQ IDNO: 1) is done by handwriting and eyeballing.89. The derivative of any of embodiments 1-88, wherein the comparison ofthe amino acid sequence of the analogue with that of GLP-1(7-37) (SEQ IDNO: 1) is done by use of a standard protein or peptide alignmentprogram.90. The derivative of embodiment 89, wherein the alignment program is aNeedleman-Wunsch alignment.91. The derivative of any of embodiments 89-90, wherein the defaultscoring matrix and the default identity matrix is used.92. The derivative of any of embodiments 89-91, wherein the scoringmatrix is BLOSUM62.93. The derivative of any of embodiments 89-92, wherein the penalty forthe first residue in a gap is −10 (minus ten).94. The derivative of any of embodiments 89-93, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).95. The derivative of any of embodiments 89-94, wherein the positioncorresponding to any of the indicated positions of GLP-1(7-37) (SEQ IDNO: 1) is identified by handwriting and eyeballing.96. The derivative of any of embodiments 89-95, wherein the positioncorresponding to any of the indicated positions of GLP-1(7-37) (SEQ IDNO: 1) is identified as described for position 26 and position 37 in anyof embodiments 61-71.97. The derivative of any of embodiments 1-96, wherein the analogue hasa maximum of four amino acid changes.98. The derivative of any of embodiments 1-97, wherein the analogue hasa maximum of three amino acid changes.99. The derivative of any of embodiments 1-98, wherein the analogue hasa maximum of two amino acid changes.100. The derivative of any of embodiments 1-99, wherein the analogue hasa maximum of one amino acid change.101. The derivative of any of embodiments 1-100, wherein the analoguehas a minimum of one amino acid modification.102. The derivative of any of embodiments 1-101, wherein the analoguehas a minimum of two amino acid changes.103. The derivative of any of embodiments 1-102, wherein the analoguehas a minimum of three amino acid changes.104. The derivative of any of embodiments 1-103, wherein the analoguehas a minimum of four amino acid changes.105. The derivative of any of embodiments 1-104, wherein the analoguehas one amino acid change.106. The derivative of any of embodiments 1-104, wherein the analoguehas two amino acid changes.107. The derivative of any of embodiments 1-104, wherein the analoguehas three amino acid changes.108. The derivative of any of embodiments 1-104, wherein the analoguehas four amino acid changes.109. The derivative of any of embodiments 1-108, wherein the changesare, independently, substitutions, additions, and/or deletions.110. The derivative of any of embodiments 1-109, wherein the changes aresubstitutions.111. The derivative of any of embodiments 1-110, wherein the analogue a)comprises a GLP-1 analogue of Formula I; and/or b) is a GLP-1 analogueof Formula I:

Formula I:Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Lys-Xaa₃₈,(SEQ ID NO: 11) wherein

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, β-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Phe or Leu;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Val, or Leu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu or Met;

Xaa₂₂ is Gly, Glu, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₅ is Ala or Val;

Xaa₂₇ is Glu or Leu;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His

Xaa₃₃ is Val;

Xaa₃₄ is Glu, Asn, Gly, Gin, Arg, or His;

Xaa₃₅ is Gly or Aib;

Xaa₃₆ is Arg or Gly; and

Xaa₃₈ is Ser, Gly, Ala, Glu, Pro, Arg, or absent.

112. The derivative of embodiment 111, wherein the peptide of Formula Iis an analogue of GLP-1(7-37) (SEQ ID NO: 1).113. The derivative of any of embodiments 111-112, wherein Xaa₇ is His;Xaa₈ is Aib; Xaa₁₂ is Phe; Xaa₁₆ is Val; Xaa₁₈ is Ser; Xaa₁₉ is Tyr;Xaa₂₀ is Leu; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gin; Xaa₂₅ is Ala; Xaa₂₇ isGlu; Xaa₃₀ is Ala; Xaa₃₁ is Trp or His; Xaa₃₃ is Val; Xaa₃₄ is Gin, Arg,or His; Xaa₃₅ is Gly; Xaa₃₆ is Arg; and Xaa₃₈ is absent.114. The derivative of any of embodiments 111-113, wherein Xaa₇ is His.115. The derivative of any of embodiments 111-114, wherein Xaa₈ is Aib.116. The derivative of any of embodiments 111-115, wherein Xaa₁₂ is Phe.117. The derivative of any of embodiments 111-116, wherein Xaa₁₆ is Val.118. The derivative of any of embodiments 111-117, wherein Xaa₁₈ is Ser.119. The derivative of any of embodiments 111-118, wherein Xaa₁₉ is Tyr.120. The derivative of any of embodiments 111-119, wherein Xaa₂₀ is Leu.121. The derivative of any of embodiments 111-120, wherein Xaa₂₂ is Gly.122. The derivative of any of embodiments 111-121, wherein Xaa₂₂ is Glu.123. The derivative of any of embodiments 111-122, wherein Xaa₂₃ is Gin.124. The derivative of any of embodiments 111-123, wherein Xaa₂₅ is Ala.125. The derivative of any of embodiments 111-124, wherein Xaa₂₇ is Glu.126. The derivative of any of embodiments 111-125, wherein Xaa₃₀ is Ala.127. The derivative of any of embodiments 111-126, wherein Xaa₃₀ is Glu.128. The derivative of any of embodiments 111-127, wherein Xaa₃₁ is Trp.129. The derivative of any of embodiments 111-128, wherein Xaa₃₁ is His.130. The derivative of any of embodiments 111-129, wherein Xaa₃₃ is Val.131. The derivative of any of embodiments 111-130, wherein Xaa₃₄ is Gin.132. The derivative of any of embodiments 111-131, wherein Xaa₃₄ is Arg.133. The derivative of any of embodiments 111-132, wherein Xaa₃₄ is His.134. The derivative of any of embodiments 111-133, wherein Xaa₃₅ is Gly.135. The derivative of any of embodiments 111-134, wherein Xaa₃₆ is Arg.136. The derivative of any of embodiments 111-135, wherein Xaa₃₈ isabsent.137. The derivative of any of embodiments 1-136, wherein the analoguecomprises the following amino acid changes, as compared to GLP-1(7-37)(SEQ ID NO: 1):(i) 8Aib, 34R, 37K; (ii) 8Aib, 34H, 37K; (iii) 8Aib, 31H, 34Q, 37K; (iv)8Aib, 34Q, 37K

(v) 8Aib, 22E, 34Q, 37K; or (vi) 8Aib, 22E, 34R, 37K.

138. The derivative of any of embodiments 1-137, wherein the analoguehas the following amino acid changes, as compared to GLP-1(7-37) (SEQ IDNO: 1):(i) 8Aib, 34R, 37K; (ii) 8Aib, 34H, 37K; (iii) 8Aib, 31H, 34Q, 37K; (iv)8Aib, 34Q, 37K

(v) 8Aib, 22E, 34Q, 37K; or (vi) 8Aib, 22E, 34R, 37K.

139. A compound, preferably according to any of embodiments 1-138,selected from the following: Chem. 21, Chem. 22, Chem. 23, Chem. 24,Chem. 25, Chem. 26, Chem. 27, Chem. 28, Chem. 29, Chem. 30, Chem. 31,Chem. 32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38,Chem. 39, Chem. 40, Chem. 41, and Chem. 42; or a pharmaceuticallyacceptable salt, amide, or ester thereof.140. A compound characterised by its name, and selected from a listingof each of the names of the compounds of Examples 1-22 herein; or apharmaceutically acceptable salt, amide, or ester thereof.141. The compound of embodiment 140, which is a compound of embodiment139.142. The derivative of any of embodiments 1-141, which has GLP-1activity.143. The derivative of embodiment 142, wherein GLP-1 activity refers tothe capability of activating the human GLP-1 receptor.144. The derivative of embodiment 143, wherein activation of the humanGLP-1 receptor is measured in an in vitro assay, as the potency of cAMPproduction.145. The derivative of any of embodiments 1-144, which has a potencycorresponding to an EC₅₀a) below 10000 pM, preferably below 8000 pM, more preferably below 5000pM, even more preferably below 4000 pM, or most preferably below 3000pM;b) below 2000 pM, preferably below 1200 pM, more preferably below 1000pM, even more preferably below 800 pM, or most preferably below 600 pM;c) below 400 pM, preferably below 300 pM, more preferably below 200 pM,even more preferably below 150 pM, or most preferably below 100 pM; ord) below 80 pM, preferably below 60 pM, more preferably below 50 pM,even more preferably below 40 pM, or most preferably below 30 pM.146. The derivative of embodiment 145, wherein the potency is determinedas EC₅₀ for stimulation of the formation of cAMP in a medium containingthe human GLP-1 receptor, preferably using a stable transfectedcell-line such as BHK467-12A (tk-ts13), and/or using for thedetermination of cAMP a functional receptor assay, e.g. based oncompetition between endogenously formed cAMP and exogenously addedbiotin-labelled cAMP, in which assay cAMP is more preferably capturedusing a specific antibody, and/or wherein an even more preferred assayis the AlphaScreen cAMP Assay, most preferably the one described inExample 44.147. The derivative of any of embodiments 1-146, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 0.001% HSA (lowalbumin) isa) below 500 nM, preferably below 250 nM, more preferably below 100 nM,or most preferably below 50 nM;b) below 10 nM, preferably below 8.0 nM, still more preferably below 6.0nM, even more preferably below 5.0 nM, or most preferably below 2.00 nM;c) below 1.00 nM, preferably below 0.80 nM, even more preferably below0.60 nM, or most preferably below 0.50 nM; ord) below 0.40 nM, preferably below 0.30 nM, even more preferably below0.20 nM, or most preferably below 0.10 nM.148. The derivative of embodiment 143, wherein activation of the humanGLP-1 receptor is measured as GLP-1 receptor binding affinity (IC₅₀) inthe presence of 0.001% HSA (low albumin).149. The derivative of any of embodiments 1-148, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 2.0% HSA (highalbumin) isa) below 1000 nM, preferably below 800 nM;b) below 700 nM, preferably below 500 nM, more preferably below 300 nM;orc) below 200 nM, preferably below 100 nM, or more preferably below 50nM.150. The derivative of any of embodiments 147-149, wherein the bindingaffinity to the GLP-1 receptor is measured by way of displacement of¹²⁵I-GLP-1 from the receptor, preferably using a SPA binding assay.151. The derivative of embodiment 150, wherein the GLP-1 receptor isprepared using a stable, transfected cell line, preferably a hamstercell line, more preferably a baby hamster kidney cell line, such as BHKtk-ts13.152. The derivative of any of embodiments 147-151, wherein the IC₅₀value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.153. The derivative of any of embodiments 1-152, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of semaglutide.154. The derivative of any of embodiments 1-153, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of liraglutide.155. The derivative of any of embodiments 1-154, wherein the derivativeis effective at lowering blood glucose in vivo in db/db mice.156. The derivative of any of embodiments 1-154, wherein the derivativeis effective at lowering body weight in vivo in db/db mice.157. The derivative of any of embodiments 1-156 which, in a PD study inpigs, reduces food intake on day 1, 2, 3, and/or 4 after s.c.administration of a single dose of the derivative, as compared to avehicle-treated control group, wherein preferably the dose is 0.3, 1, 3,10 or 30 nmol/kg; more preferably 3.0 nmol/kg.158. The derivative of embodiment 157, wherein the food intake on day 1is reduced to 80% or lower, preferably to 60% or lower, more preferablyto 50% or lower, or most preferably to 15% or lower.159. The derivative of any of embodiments 157-158, wherein the foodintake on day 2 is reduced to 80% or lower, preferably to 60% or lower,more preferably to 40% or lower, or most preferably to 20% or lower.160. The derivative of any of embodiment 157-159, wherein the foodintake on day 3 is reduced to 90% or lower, preferably to 80% or lower,or most preferably to 75% or lower.161. The derivative of any of embodiments 157-160, wherein the study isconducted and the data compiled and analysed as described in Example 49.162. The derivative of any of embodiments 1-161, which has a moreprotracted profile of action than liraglutide.163. The derivative of embodiment 162, wherein protraction meanshalf-life in vivo in a relevant animal species, such as db/db mice, rat,pig, and/or, preferably, minipig; wherein the derivative is administeredi) s.c., and/or, ii) i.v.; preferably ii) i.v.164. The derivative of any of embodiments 1-163, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is higher than thatof semaglutide.165. The derivative of any of embodiments 1-164, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least a) 25%higher, b) 50% higher, c) 75% higher, or d) 100% higher (=twice) thanthe terminal half-life of semaglutide.166. The derivative of any of embodiments 1-165, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least threetimes the terminal half-life of semaglutide.167. The derivative of any of embodiments 1-166, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least fourtimes the terminal half-life of semaglutide.168. The derivative of any of embodiments 1-167, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in rat is at least fivetimes the terminal half-life of semaglutide.169. The derivative of any of embodiments 163-168, wherein the half-lifeis determined in in vivo pharmacokinetic studies in rat, for example asdescribed in Example 48.170. The derivative of any of embodiments 1-169, wherein the terminalhalf-life (T_(1/2)) after i.v. administration in minipigs isa) at least 8 hours, preferably at least 16 hours, more preferably atleast 24 hours, even more preferably at least 32 hours, or mostpreferably at least 40 hours;b) at least 50 hours, preferably at least 60 hours, more preferably atleast 70 hours, even more preferably at least 80 hours, or mostpreferably at least 90 hours; orc) at least 95 hours, preferably at least 100 hours.171. The derivative of embodiment 170, wherein the minipigs are maleGöttingen minipigs.172. The derivative of any of embodiments 170-171, wherein the minipigsare 7-14 months of age, and preferably weighing from 16-35 kg.173. The derivative of any of embodiments 170-172, wherein the minipigsare housed individually, and fed once or twice daily, preferably withSDS minipig diet.174. The derivative of any of embodiments 170-173, wherein thederivative is dosed, i.v., after at least 2 weeks of acclimatisation.175. The derivative of any of embodiments 170-174, wherein the animalsare fasted for approximately 18 h before dosing and from 0 to 4 h afterdosing, and have ad libitum access to water during the whole period.176. The derivative of any of embodiments 170-175, wherein the GLP-1derivative is dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a suitable concentration, preferablyfrom 20-60 nmol/ml.177. The derivative of any of embodiments 170-176, wherein intravenousinjections of the derivative are given in a volume corresponding to 1-2nmol/kg.178. An intermediate product in the form of a GLP-1 analogue whichcomprises the following changes as compared to GLP-1(7-37) (SEQ ID NO:1): (ii) 8Aib, 34H, 37K; or (v) 8Aib, 22E, 34Q, 37K; or apharmaceutically acceptable salt, amide, or ester of any of theanalogues of (ii) or (v).179. An intermediate product in the form of a GLP-1 analogue selectedfrom the following analogues of GLP-1(7-37) (SEQ ID NO: 1): (ii) 8Aib,34H, 37K; (v) 8Aib, 22E, 34Q, 37K; and (vi) 8Aib, 22E, 34R, 37K; or apharmaceutically acceptable salt, amide, or ester of any of theanalogues of (ii), (v), or (vi).180. The analogue of any of embodiments 178-179, wherein the comparisonwith GLP-1(7-37) (SEQ ID NO: 1) is made by handwriting and eyeballing.181. The analogue of any of embodiments 178-180, wherein the comparisonwith GLP-1(7-37) (SEQ ID NO: 1) is made by use of a standard protein orpeptide alignment program.182. The analogue of embodiment 181, wherein the alignment program is aNeedleman-Wunsch alignment.183. The analogue of any of embodiments 181-182, wherein the defaultscoring matrix and the default identity matrix is used.184. The analogue of any of embodiments 181-183, wherein the scoringmatrix is BLOSUM62.185. The analogue of any of embodiments 181-184, wherein the penalty forthe first residue in a gap is −10 (minus ten).186. The analogue of any of embodiments 181-185, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).187. A pharmaceutical composition comprising a derivative according toany of embodiments 1-186, and a pharmaceutically acceptable excipient.188. A derivative according to any of embodiments 1-186, for use as amedicament.189. A derivative according to any of embodiments 1-186, for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.190. Use of a derivative according to any of embodiments 1-186 in themanufacture of a medicament for the treatment and/or prevention of allforms of diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.191. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of embodiments 1-186.

The invention also relates to

a). A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 26 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), anda maximum of eight amino acid changes as compared to GLP-1(7-37);

which derivative comprises two protracting moieties attached to thefirst and second K residue, respectively, via a linker, wherein theprotracting moiety is selected from Chem. 2, and Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

HOOC—(CH₂)_(x)—CO—*,  Chem. 1:

-   -   in which x is an integer in the range of 8-14, y is an integer        in the range of 6-13, and the linker comprises Chem. 3:

*—NH—(CH₂)_(q)—CH[(CH₂)_(w)—NH₂]—CO—*,  Chem. 3:

which is connected at its CO—* end to the epsilon amino group of thefirst or the second K residue of the GLP-1 analogue, and wherein q is aninteger in the range of 0-5, and w is an integer in the range of 0-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

b). The derivative of embodiment 1, wherein the linker comprises z timesChem. 3, wherein z is an integer in the range of 1-2.c). The derivative of any of embodiments 1-2, wherein w is 0.d). The derivative of any of embodiments 1-3, wherein q is 4.e). The derivative of any of embodiments 1-4, wherein the linkercomprises

*—NH—(CH₂)_(q)—CH(NH₂)—CO—*,  Chem. 4:

wherein q is an integer in the range of β-5.f). The derivative of any of embodiments 1-6, wherein the linkercomprises

*—NH—(CH₂)₄—CH(NH₂)—CO—*.  Chem. 6:

g). The derivative of any of embodiments 1-6, wherein x is 12 or 14.h). The derivative of any of embodiments 1-6, wherein y is 9, 10, or 11.i). The derivative of any of embodiments 1-8, wherein the analoguecomprises a GLP-1 analogue of Formula I:

Formula I:Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Lys-Xaa₃₈,(SEQ ID NO: 11) wherein

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, β-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Phe or Leu;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Val, or Leu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu or Met;

Xaa₂₂ is Gly, Glu, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₅ is Ala or Val;

Xaa₂₇ is Glu or Leu;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His

Xaa₃₃ is Val;

Xaa₃₄ is Glu, Asn, Gly, Gin, Arg, or His;

Xaa₃₅ is Gly or Aib;

Xaa₃₆ is Arg or Gly; and

Xaa₃₈ is Ser, Gly, Ala, Glu, Pro, Arg, or absent.

j). A compound selected from the following: Chem. 21, Chem. 22, Chem.23, Chem. 24, Chem. 25, Chem. 26, Chem. 27, Chem. 28, Chem. 29, Chem.30, Chem. 31, Chem. 32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem.37, Chem. 38, Chem. 39, Chem. 40, Chem. 41, and Chem. 42; or apharmaceutically acceptable salt, amide, or ester thereof.k). A pharmaceutical composition comprising a derivative according toany of embodiments 1-10, and a pharmaceutically acceptable excipient.l). A derivative according to any of embodiments 1-10, for use as amedicament.m). A derivative according to any of embodiments 1-10, for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.n). Use of a derivative according to any of embodiments 1-10 in themanufacture of a medicament for the treatment and/or prevention of allforms of diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.o). A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of embodiments 1-10.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods for synthesising andcharacterising analogues and derivatives of the invention. Then followsa number of examples which relate to the preparation of specific GLP-1derivatives, and at the end a number of examples have been includedrelating to the activity and properties of these analogues andderivatives (section headed pharmacological methods).

The examples serve to illustrate the invention.

LIST OF ABBREVIATIONS

-   Aib: α-aminoisobutyric acid-   AcOH: acetic acid-   API: Active Pharmaceutical Ingredient-   AUC: Area Under the Curve-   BG: Blood Glucose-   BHK Baby Hamster Kidney-   BW: Body Weight-   Boc: t-butyloxycarbonyl-   Bom: benzyloxymethyl-   BSA: Bovine serum albumin-   Bzl: benzyl-   CAS: Chemical Abstracts Service-   Clt: 2-chlorotrityl-   collidine: 2,4,6-trimethylpyridine-   DCM: dichloromethane-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-   DesH: des-amino histidine (may also be referred to as    imidazopropionic acid, Imp)-   DIC: diisopropylcarbodiimide-   DIPEA: diisopropylethylamine-   DMEM: Dulbecco's Modified Eagle's Medium (DMEM)-   EDTA: ethylenediaminetetraacetic acid-   EGTA: ethylene glycol tetraacetic acid-   FCS: Fetal Calf Serum-   Fmoc: 9-fluorenylmethyloxycarbonyl-   HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluoro-phosphate)-   HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate)-   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol-   HOAt: 1-hydroxy-7-azabenzotriazole-   HOBt: 1-hydroxybenzotriazole-   HPLC: High Performance Liquid Chromatography-   HSA: Human Serum Albumin-   IBMX: 3-isobutyl-1-methylxanthine-   Imp: Imidazopropionic acid (also referred to as des-amino histidine,    DesH)-   i.v. intravenously-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-β-methylbutyl-   IVGTT: Intravenous Glucose Tolerance Test-   LCMS: Liquid Chromatography Mass Spectroscopy-   LYD: Landrace Yorkshire Duroc-   MALDI-MS: See MALDI-TOF MS-   MALDI-TOF MS: Matrix-Assisted Laser Desorption/Ionisation Time of    Flight Mass Spectroscopy-   MeOH: methanol-   Mmt: 4-methoxytrityl-   Mtt: 4-methyltrityl-   NMP: N-methyl pyrrolidone-   OBz: benzoyl ester-   OEG: 8-amino-3,6-dioxaoctanic acid-   OPfp: pentafluorophenoxy-   OPnp: para-nitrophenoxy-   OSu: O-succinimidyl esters (hydroxysuccinimide esters)-   OtBu: tert butyl ester-   Oxyma Pure®: Cyano-hydroxyimino-acetic acid ethyl ester-   Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-   PBS: Phosphate Buffered Saline-   PD: Pharmacodynamic-   Pen/Strep: Pencillin/Streptomycin-   PK: Pharmacokinetic-   RP: Reverse Phase-   RP-HPLC: Reverse Phase High Performance Liquid Chromatography-   RT: Room Temperature-   Rt: Retention time-   s.c.: Subcutaneously-   SD: Standard Deviation-   SEC-HPLC: Size Exclusion High Performance Liquic Chromatography-   SEM: Standard Error of Mean-   SPA: Scintillation Proximity Assay-   SPPS: Solid Phase Peptide Synthesis-   tBu: tert. butyl-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane-   TLC: Thin Layer Chromatography-   Tos: tosylate (or pare-toluenesulfonyl)-   Tris: tris(hydroxymethyl)aminomethane or    2-amino-2-hydroxymethyl-propane-1,3-diol-   Trt: triphenylmethyl (trityl)-   Trx: tranexamic acid-   UPLC: Ultra Performance Liquid Chromatography

Materials and Methods Materials

α-picoline borane complex (CAS 3999-38-0)Cyano-hydroxyimino-acetic acid ethyl ester (CAS 3849-21-6)

N-α,N-β-Di-Fmoc-L-2,3-Diaminopropionic Acid (CAS 201473-90-7)

3,5-Di-tert-butyl-4-hydroxybenzoic acid (CAS 1421-49-4)3,5-Di-tert-butylbenzoic Acid (CAS 16225-26-6)Fmoc-8-amino-3,6-dioxaoctanoic acid (CAS 166108-71-0)17-(9-Fluorenylmethyloxycarbonyl-amino)-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoicacid (IRIS Biotech GmbH)Fmoc-L-Glutamic acid 1-tert-butyl ester (CAS 84793-07-7)2-(2-Methoxyethoxy)acetic acid (CAS 16024-56-9)N-α,N-ε-Bis(9-fluorenylmethyloxycarbonyl)-L-lysine (CAS 78081-87-5)1-[(9H-fluoren-9-ylmethoxy)carbonyl]piperidine-4-carboxylic acid (CAS148928-15-8)FMOC-8-Aminocapryl acid (CAS 126631-93-4)4-Phenylbutyric acid (CAS 1716-12-7)4-(4-Nitrophenyl)butyric acid (CAS 5600-62-4)4-(4-Chlorophenyl)butyric acid (CAS 4619-18-5)FMOC-6-Aminohexanoic acid (CAS 88574-06-5)FMOC-12-Aminododecanoic acid (CAS 128917-74-8)4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 1 and 2 of WO 2006/082204)4-(8-Carboxy-octyloxy)-benzoic acid tert-butyl ester (M.p.: 71-72° C.

¹H NMR (300 MHz, CDCl₃, 8H): 7.93 (d, J=8.9 Hz, 2H); 6.88 (d, J=8.9 Hz,2H); 4.00 (t, J=6.4 Hz, 2H); 2.36 (t, J=7.4 Hz, 2H); 1.80 (m, 2H); 1.65(m, 2H); 1.59 (s, 9H); 1.53-1.30 (m, 8H) (prepared as described inExample 25, step 1 and 2 of WO 2006/082204, replacing methyl10-bromodecanoate with ethyl 9-Bromononanoate (CAS 28598-81-4))4-(7-Carboxy-heptyloxy)-benzoic acid tert-butyl ester (¹H NMR spectrum(300 MHz, CDCl₃, δ_(H)): 7.93 (d, J=9.0 Hz, 2H); 6.88 (d, J=9.0 Hz, 2H);4.00 (t, J=6.5 Hz, 2H); 2.37 (t, J=7.4 Hz, 2H); 1.80 (m, 2H); 1.64 (m,2H); 1.59 (s, 9H); 1.53-1.33 (m, 6H)) (prepared as described in Example25, step 1 and 2 of WO 2006/082204, replacing methyl 10-bromodecanoatewith ethyl 7-bromoheptanoate (CAS 29823-18-5))

Chemical Methods

This section is divided in two: Section A relating to general methods(of preparation (A1); and of detection and characterisation (A2)), andsection B, in which the preparation and characterisation of a number ofspecific example compounds is described.

A. GENERAL METHODS A1. Methods of Preparation

This section relates to methods for solid phase peptide synthesis (SPPSmethods, including methods for de-protection of amino acids, methods forcleaving the peptide from the resin, and for its purification), as wellas methods for detecting and characterising the resulting peptide (LCMS,MALDI, and UPLC methods). The solid phase synthesis of peptides may insome cases be improved by the use of di-peptides protected on thedi-peptide amide bond with a group that can be cleaved under acidicconditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or2,4,6-trimethoxybenzyl. In cases where a serine or a threonine ispresent in the peptide, pseudoproline di-peptides may be used (availablefrom, e.g., Novabiochem, see also W. R. Sampson (1999), J. Pep. Sci. 5,403). The Fmoc-protected amino acid derivatives used were the standardrecommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH,or, Fmoc-Val-OH etc. supplied from e.g. Anaspec, Bachem, Iris Biotech,or Novabiochem. Where nothing else is specified the natural L-form ofthe amino acids are used. The N-terminal amino acid was Boc protected atthe alpha amino group (e.g. Boc-His(Boc)-OH, or Boc-His(Trt)-OH forpeptides with His at the N-terminus). In case of modular albumin bindingmoiety attachment using SPPS the following suitably protected buildingblocks such as but not limited to Fmoc-8-amino-3,6-dioxaoctanoic acid,Fmoc-tranexamic acid, Fmoc-Glu-OtBu, octadecanedioic acidmono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester,tetradecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were used. All operations stated belowwere performed at 250-μmol synthesis scale.

1. Synthesis of Resin Bound Protected Peptide Backbone Method: SPPS_P

SPPS_P was performed on a Prelude Solid Phase Peptide Synthesiser fromProtein Technologies (Tucson, Ariz. 85714 U.S.A.) at 250-μmol scaleusing six fold excess of Fmoc-amino acids (300 mM in NMP with 300 mMHOAt or Oxyma Pure®) relative to resin loading, e.g. low loadFmoc-Gly-Wang (0.35 mmol/g). Fmoc-deprotection was performed using 20%piperidine in NMP. Coupling was performed using 3:3:3:4 amino acid/(HOAtor Oxyma Pure®)/DIC/collidine in NMP. NMP and DCM top washes (7 ml, 0.5min, 2×2 each) were performed between deprotection and coupling steps.Coupling times were generally 60 minutes. Some amino acids including,but not limited to Fmoc-Arg(Pbf)-OH, Fmoc-Aib-OH or Boc-His(Trt)-OH were“double coupled”, meaning that after the first coupling (e.g. 60 min),the resin is drained and more reagents are added (amino acid, (HOAt orOxyma Pure®), DIC, and collidine), and the mixture allowed to reactagain (e.g. 60 min).

Method: SPPS_L

SPPS_L was performed on a microwave-based Liberty peptide synthesiserfrom CEM Corp. (Matthews, N.C. 28106, U.S.A.) at 250-μmol or 100-μmolscale using six fold excess of Fmoc-amino acids (300 mM in NMP with 300mM HOAt or Oxyma Pure®) relative to resin loading, e.g. low loadFmoc-Gly-Wang (0.35 mmol/g). Fmoc-deprotection was performed using 5%piperidine in NMP at up to 75° C. for 30 seconds where after the resinwas drained and washed with NMP and the Fmoc-deprotection was repeatedthis time for 2 minutes at 75° C. Coupling was performed using 1:1:1amino acid/(HOAt or Oxyma Pure®)/DIC in NMP. Coupling times andtemperatures were generally 5 minutes at up to 75° C. Longer couplingtimes were used for larger scale reactions, for example 10 min.Histidine amino acids were double coupled at 50° C., or quadruplecoupled if the previous amino acid was sterically hindered (e.g. Aib).Arginine amino acids were coupled at RT for 25 minutes and then heatedto 75° C. for 5 min. Some amino acids such as but not limited to Aib,were “double coupled”, meaning that after the first coupling (e.g. 5 minat 75° C.), the resin is drained and more reagents are added (aminoacid, (HOAt or Oxyma Pure®) and DIC), and the mixture is heated again(e.g. 5 min at 75° C.). NMP washes (5×10 ml) were performed betweendeprotection and coupling steps.

Method: SPPS_A

The protected peptidyl resin was synthesised according to the Fmocstrategy on an Applied Biosystems 433 peptide synthesiser in a 250-μmolor 1000 μmol scale with three or four fold excess of Fmoc-amino acids,using the manufacturer supplied FastMoc UV protocols which employ HBTU(2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate) or HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) mediated couplings in NMP and UV monitoring of thedeprotection of the Fmoc protection group, in some cases doublecouplings were used, meaning that after the first coupling, the resin isdrained and more Fmoc-amino acids and reagents are added. The startingresin used for the synthesis of the peptide amides was Rink-Amide resinand either preloaded Wang (e.g. low load Fmoc-Gly-Wang orFmoc-Lys(Mtt)-wang) or chlorotrityl resin for peptides with a carboxyC-terminal. The protected amino acid derivatives used were standardFmoc-amino acids (supplied from e.g. Anaspec, or Novabiochem) suppliedin preweighed cartridges suitable for the ABI433A synthesiser with theexception of unnatural aminoacids such as Fmoc-Aib-OH(Fmoc-aminoisobutyric acid). The N terminal amino acid was Boc protectedat the alpha amino group (e.g. Boc-His(Boc)-OH or Boc-His(Trt)-OH wasused for peptides with His at the N-terminal). The epsilon amino groupof lysines in the sequence were either protected with Mtt, Mmt, Dde,ivDde, or Boc, depending on the route for attachment of the albuminbinding moiety and spacer. The synthesis of the peptides may in somecases be improved by the use of dipeptides protected on the dipeptideamide bond with a group that can be cleaved under acidic conditions suchbut not limited to 2-Fmoc-oxy-4-methoxybenzyl or 2,4,6-trimethoxybenzyl.In cases where a serine or a threonine is present in the peptide, theuse of pseudoproline dipeptides may be used (see e.g. catalogue fromNovobiochem 2009/2010 or newer version, or W. R. Sampson (1999), J. Pep.Sci. 5, 403).

Method: SPPS_M

SPPS_M refers to synthesis of the protected peptidyl resin using manualFmoc chemistry. The coupling chemistry was DIC/(HOAt or OxymaPure®)/collidine in NMP at a 4-10 fold molar excess. Coupling conditionswere 1-6 h at room temperature. Fmoc-deprotection was performed with20-25% piperidine in NMP (3×20 ml, each 10 min) followed by NMP washings(4×20 mL).

2. Synthesis of Side Chains Mono Esters of Fatty Diacids

Overnight reflux of the C8, C10, C12, C14, C16 and C18 diacids withBoc-anhydride DMAP t-butanol in toluene gives predominately the t-butylmono ester. Obtained is after work-up a mixture of mono acid, diacid anddiester. Purification is carried out by washing, short plug silicafiltration and crystallisation.

3. Attachment of Side Chains to Resin Bound Protected Peptide Backbone

When an acylation is present on a lysine side chain, the epsilon aminogroup of lysine to be acylated was protected with either Mtt, Mmt, Dde,ivDde, or Boc, depending on the route for attachment of the protractingmoiety and linker. Dde- or ivDde-deprotection was performed with 2%hydrazine in NMP (2×20 ml, each 10 min) followed by NMP washings (4×20ml). Mtt- or Mmt-deprotection was performed with 2% TFA and 2-3% TIS inDCM (5×20 ml, each 10 min) followed by DCM (2×20 ml), 10% MeOH and 5%DIPEA in DCM (2×20 ml) and NMP (4×20 ml) washings, or by treatment withhexafluoroisopropanol/DCM (75:25, 5×20 ml, each 10 min) followed bywashings as above. In some cases the Mtt group was removed by automatedsteps on the Liberty peptide synthesiser. Mtt deprotection was performedwith hexafluoroisopropanol or hexafluoroisopropanol/DCM (75:25) at roomtemperature for 30 min followed by washing with DCM (7 ml×5), followedby NMP washings (7 ml×5). The protracting moiety and/or linker can beattached to the peptide either by acylation of the resin bound peptideor by acylation in solution of the unprotected peptide. In case ofattachment of the protracting moiety and/or linker to the protectedpeptidyl resin the attachment can be modular using SPPS and suitablyprotected building blocks.

Method: SC_P

The N-ε-lysine protection group was removed as described above and thechemical modification of the lysine was performed by one or moreautomated steps on the Prelude peptide synthesiser using suitablyprotected building blocks as described above. Double couplings wereperformed as described in SPPS_P with 3 hours per coupling.

Method: SC_L

The N-ε-lysine protection group was removed as described above and thechemical modification of the lysine was performed by one or moreautomated steps on the Liberty peptide synthesiser using suitablyprotected building blocks as described above. Double couplings wereperformed as described in SPPS_L.

Method: SC_A

The N-ε-lysine protection group was removed as described above and thechemical modification of the lysine was performed by one or moreautomated steps on the ABI peptide synthesiser using suitably protectedbuilding blocks as described in SPPS_A.

Method: SC_M1

The N-ε-lysine protection group was removed as described above.Activated (active ester or symmetric anhydride) protracting moiety orlinker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600, 4 molar equivalents relative to resinbound peptide) was dissolved in NMP (25 mL), added to the resin andshaken overnight at room temperature. The reaction mixture was filteredand the resin was washed extensively with NMP, DCM, 2-propanol, methanoland diethyl ether.

Method: SC_M2

The N-ε-lysine protection group was removed as described above. Theprotracting moiety was dissolved in NMP/DCM (1:1, 10 ml). The activatingreagent such as HOBt or Oxyma Pure® (4 molar equivalents relative toresin) and DIC (4 molar equivalents relative to resin) was added and thesolution was stirred for 15 min. The solution was added to the resin andDIPEA (4 molar equivalents relative to resin) was added. The resin wasshaken 2 to 24 hours at room temperature. The resin was washed with NMP(2×20 ml), NMP/DCM (1:1, 2×20 ml) and DCM (2×20 ml).

Method: SC_M3

Activated (active ester or symmetric anhydride) protracting moiety orlinker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5 molar equivalents relative to thepeptide was dissolved in an organic solvent such as acetonitrile, THF,DMF, DMSO or in a mixture of water/organic solvent (1-2 ml) and added toa solution of the peptide in water (10-20 ml) together with 10 molarequivalents of DIPEA. In case of protecting groups on the protractingmoiety such as tert-butyl, the reaction mixture was lyophilisedovernight and the isolated crude peptide deprotected afterwards. In caseof tert-butyl protection groups the deprotection was performed bydissolving the peptide in a mixture of trifluoroacetic acid, water andtriisopropylsilane (90:5:5). After 30 min the mixture was evaporated invacuo and the crude peptide purified by preparative HPLC as describedlater.

4. Clevage of Resin Bound Peptide with or without Attached Side Chainsand Purification

Method: CP_M1

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by a 2-3 hour treatment with TFA/TIS/water(95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.The peptide was dissolved in a suitable solvent (such as, e.g., 30%acetic acid) and purified by standard RP-HPLC on a C18, 5 M column,using acetonitrile/water/TFA. The fractions were analysed by acombination of UPLC, MALDI and LCMS methods, and the appropriatefractions were pooled and lyophilised.

Method: CP_L1

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by the use of a CEM Accent Microwave CleavageSystem (CEM Corp., North Carolina). Cleavage from the resin wasperformed at 38° C. for 30 minutes by the treatment with TFA/TIS/water(95/2.5/2.5) followed by precipitation with diethylether. The peptidewas dissolved in a suitable solvent (such as, e.g., 30% acetic acid) andpurified by standard RP-HPLC on a C18, 5 M column, usingacetonitrile/water/TFA. The fractions were analysed by a combination ofUPLC, MALDI and LCMS methods, and the appropriate fractions were pooledand lyophilised.

A2. General Methods for Detection and Characterisation 1. LC-MS MethodsMethod: LCMS01v1

LCMS01v1 was performed on a setup consisting of Waters Acquity UPLCsystem and LCT Premier XE mass spectrometer from Micromass. Eluents: A:0.1% Formic acid in water

B: 0.1% Formic acid in acetonitrile The analysis was performed at RT byinjecting an appropriate volume of the sample (preferably 2-10 μl) ontothe column which was eluted with a gradient of A and B. The UPLCconditions, detector settings and mass spectrometer settings were:Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm×50 mm. Gradient:Linear 5%-95% acetonitrile during 4.0 min (alternatively 8.0 min) at 0.4ml/min. Detection: 214 nm (analogue output from TUV (Tunable UVdetector)) MS ionisation mode: API-ES Scan: 100-2000 amu (alternatively500-2000 amu), step 0.1 amu.

Method: LCMS 4

LCMS_4 was performed on a setup consisting of Waters Acquity UPLC systemand LCT Premier XE mass spectrometer from Micromass. Eluents: A: 0.1%Formic acid in water B: 0.1% Formic acid in acetonitrile The analysiswas performed at RT by injecting an appropriate volume of the sample(preferably 2-10 μl) onto the column which was eluted with a gradient ofA and B. The UPLC conditions, detector settings and mass spectrometersettings were: Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm×50mm. Gradient: Linear 5%-95% acetonitrile during 4.0 min (alternatively8.0 min) at 0.4 ml/min. Detection: 214 nm (analogue output from TUV(Tunable UV detector)) MS ionisation mode: API-ES Scan: 100-2000 amu(alternatively 500-2000 amu), step 0.1 amu.

Method: LCMS_AP

LCMS_AP was performed using a Micromass Quatro micro API massspectrometer to identify the mass of the sample after elution from aHPLC system composed of Waters 2525 binary gradient modul, Waters 2767sample manager, Waters 2996 Photodiode Array Detector and Waters 2420ELS Detector. Eluents: A: 0.1% Trifluoro acetic acid in water; B: 0.1%Trifluoro acetic acid in acetonitrile. Column: Phenomenex Synergi MAXRP,4 um, 75×4.6 mm. Gradient: 5%-95% B over 7 min at 1.0 ml/min.

2. UPLC Methods Method: UPLC02v1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,40° C. The UPLC system was connected to two eluent reservoirscontaining: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05% TFA. Thefollowing linear gradient was used: 95% A, 5% B to 95% A, 5% B over 16minutes at a flow-rate of 0.40 ml/min.

Method: UPLC07v1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 215 nm and 254 nm were collectedusing an kinetex 1.7u C18, 100A 2.1×150 mm column, 60° C. The UPLCsystem was connected to two eluent reservoirs containing: A: 90% waterand 10% CH₃CN with 0.045M (NH₄)₂HPO₄, pH 3.6, B: 20% isopropanole, 20%water and 60% CH₃CN. The following step gradient was used: 35% B and 65%A over 2 minutes, then 35% B, 65% A to 65% B, 35% A over 15 minutes,then 65% B, 35% A to 80% B, 20% A over 3 minutes at a flowrate of 0.5ml/min.

Method: UPLC06v1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 215 nm and 254 nm were collectedusing an kinetex 1.7u C18, 100A 2.1×150 mm column, 60° C. The UPLCsystem was connected to two eluent reservoirs containing: A: 90% waterand 10% MeCN with 0.045M (NH₄)₂HPO₄, pH 3.6, B: 20% isopropanole, 20%water and 60% CH₃CN. The following step gradient was used: 25% B and 75%A over 2 minutes, then 25% B, 75% A to 55% B, 45% A over 15 minutes,then 55% B, 45% A to 80% B, 20% A over 3 minutes at a flowrate of 0.5ml/min.

Method: AP_B4_1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,30° C. The UPLC system was connected to two eluent reservoirscontaining: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05% TFA. Thefollowing linear gradient was used: 95% A, 5% B to 5% A, 95% B over 16minutes at a flow-rate of 0.30 ml/min.

Method: B4_1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,40° C. The UPLC system was connected to two eluent reservoirscontaining: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05% TFA. Thefollowing linear gradient was used: 95% A, 5% B to 5% A, 95% B over 16minutes at a flow-rate of 0.40 ml/min.

Method: B31_1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 215 nm and 254 nm were collectedusing a kinetex column 1.7u C18, 100A 2.1×150 mm column, 60° C. The UPLCsystem was connected to two eluent reservoirs containing: A: 90% waterand 10% MeCN with 0.045M (NH₄)₂HPO₄, pH 3.6, B: 20% isopropanole, 20%water and 60% CH₃CN. The following step gradient was used: 25% B and 75%A over 2 minutes, then 25% B, 75% A to 55% B, 45% A over 15 minutes,then 55% B, 45% A to 80% B, 20% A over 3 minutes at a flowrate of 0.5ml/min.

Method: UPLC22v1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,40° C. The UPLC system was connected to two eluent reservoirscontaining: A: 10 mM TRIS, 15 mM ammonium sulphate, 80% H₂O, 20% CH₃CN,pH 7.3; B: 80% CH₃CN, 20% H₂O. The following linear gradient was used:95% A, 5% B to 10% A, 90% B over 16 minutes at a flow-rate of 0.35ml/min.

3. MALDI-MS Method Method: MALDI01v1

Molecular weights were determined using matrix-assisted laser desorptionand ionisation time-of-flight mass spectroscopy, recorded on a Microflexor Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acidwas used.

B. EXAMPLE COMPOUNDS Example 1N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4820. found m/z: 4817.UPLC method: UPLC06v1: Rt=13.7 min

Example 2N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1UPLC method: UPLC02v1: Rt=7.8 minUPLC method: UPLC06v1: Rt=13.7 min

Example 3N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,His³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 4.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4701.4. found m/z: 4699.6.UPLC method: UPLC02v1: Rt=7.7 minUPLC method: UPLC06v1: Rt=13.9 min

Example 4N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 5.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4799.5. found m/z: 4796.UPLC method: UPLC06v1: Rt=15.1 minUPLC method: UPLC02v1: Rt=7.8 min

Example 5N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3Preparation method: SPPS_A; SC_A; CP_M1LCMS method: LCMS01v1: Rt=2.0 min; m/4=1287; m/5=1030; m/6=858UPLC method: UPLC02v1: Rt=8.1 min

Example 6N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4776.6. found m/z: 4776.UPLC method: UPLC02v1: Rt=8.3 minUPLC method: UPLC07v1: Rt=12.0 min

Example 7N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1 calc. m/z: 4876.7. found m/z: 4875.4.UPLC method: UPLCH2v1: Rt=8.4 minUPLC method: UPLC07v1: Rt=11.9 min

Example 8N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 5080. found m/z: 5080.UPLC method: UPLC06v1: Rt=14.3 minUPLC method: UPLC02v1: Rt=8.8 min

Example 9N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 6.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4848. found m/z: 4847.UPLC method: UPLC02v1: Rt=8.4 minUPLC method: UPLC07v1: Rt=12.8 min

Example 10N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt=2.27 min; m/3=1702; m/4=1276; m/5=1021UPLC method: UPLC02v01: Rt=8.4 minUPLC method: UPLC07v01: Rt=12.9 min

Example 11 N^(ε26)-[(2S)-2-am ino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt=2.27 min; m/4=1268; m/5=1015; m/6=846UPLC method: UPLC02v1: Rt=8.42 minUPLC method: UPLC07v1: Rt=12.93 min

Example 12N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt: 2.20 min; m/3=1679; m/4=1260; m/5=1008;m/6=840UPLC method: UPLC02v1: Rt=8.00 minUPLC method: UPLC07v1: Rt=12.16 min

Example 13N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt=2.34 min; m/3=1680; m/4=1260; m/5=1008;m/6=840UPLC method: UPLC02v1: Rt=8.40 minUPLC method: UPLC07v1: Rt=13.13 min

Example 14N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt=2.27 min; m/3=1735; m/4=1301; m/5=1041;m/6=868UPLC method: UPLC07v1: Rt=12.87 minUPLC method: UPLC02v1: Rt=8.72 min

Example 15N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt: 2.24 min; m/3=1724; m/4=1293; m/5=1035;m/6=862UPLC method: UPLC02v1: Rt=8.38 minUPLC method: UPLC07v1: Rt=12.87 min

Example 16N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt=2.27 min; m/4=1285; m/5=1027; m/6=857UPLC method: UPLC02v1: Rt=8.5 minUPLC method: UPLC07v1: Rt=11.4 min

Example 17N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_L; SC_M1; CP_M1LCMS method: LCMS01v1: Rt: 2.32 min; m/4=1285; m/5=1028UPLC method: UPLC02v1: Rt=8.28 minUPLC method: UPLC07v1: Rt=12.52 min

Example 18N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Glu²²,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 7.Preparation method: SPPS_A; SC_A; CP_M1LCMS method: LCMS1v1: Rt=2.2 min; m/3=1731; m/4=1298; m/5=1039UPLC method: UPLC02v1: Rt=8.2 min

Example 19N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where amino acid sequence is that of SEQ ID NO: 3.Preparation method SPPS_P; SC_P; CP_M1MALDI method: MALDI01v: calc. m/z: 5044.8. found m/z: 5042.5.UPLC method: UPLC02v1: Rt=7.8 minUPLC method: UPLC07v1: Rt=9.8 min

Example 20N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where there the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 4848. found m/z: 4845.UPLC method: UPLC02v1: Rt=8.0 minUPLC method: UPLC07v1: Rt=10.8 min

Example 21N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Glu²²,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 8Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI01v1: calc. m/z: 5273; found m/z: 5272.UPLC method: UPLC02v1: Rt=8.7 minUPLC method: UPLC07v1: Rt=12.4 min

Example 22N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbexyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Glu²²,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 8.Preparation method: SPPS_P; SC_P; CP_M1MALDI method: MALDI0v1: calc. m/z: 5244.9; found m/z: 5243.4.UPLC method: UPLC02v1: Rt=8.2 minUPLC method: UPLC07v1: Rt=11.1 min

Example 23N^(ε26)-[(2S)-2-amino-6-[[2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_A (alternatively SPPS_L), SC_A, CP_M1

UPLC Method: B4_1: Rt=8.70 min

LCMS Method: LCMS_4: Rt=2.10 min; m/3:1849; m/4:1387; m/5:1110

Example 24N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1

UPLC Method: AP_B4_1: Rt=8.4 min

LCMS Method: LCMS_4: Rt=2.15 min, m/3=1695; m/4=1271

Example 25N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]-[Imp⁷,Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 9.Preparation method: SPPS_P; SC_P; CP_M1

UPLC Method: AP_B4_1: Rt=8.5 min

LCMS Method: LCMS_4: Rt=2.2 min, m/3=1690; m/4=1268

Example 26N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[8-[10-(4-carboxyphenoxy)decanoylamino]octanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[8-[10-(4-carboxyphenoxy)decanoylamino]octanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI-MS method: MALDI01v01: calc. m/z: 5097, found m/z: 5101.

UPLC Method: B31_1: Rt=16.5 min Example 27N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1MALDI-MS method: MALDI01v01: calc. m/z: 4832, found m/z: 4832.

UPLC Method: B4_1: Rt=8.8 min Example 28N^(ε26)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib⁸,Glu²²,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 7.Preparation method: SPPS_A; SC_A; CP_M1LCMS Method: LCMS_4: Rt=2.43 min, m/3=1735; m/4=1301

UPLC Method: B4_1: Rt=9.52 min Example 29N^(ε26)-[(2S)-6-[[(2S)-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-2-(dimethylamino)hexanoyl]amino]-2-(dimethylamino)hexanoyl],N^(ε37)-[(2S)-6-[[(2S)-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-2-(dimethylamino)hexanoyl]amino]-2-(dimethylamino)hexanoyl]-[Imp⁷,Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 9.Preparation method: SPPS_P; SC_P; CP_M1 (using the intermediate compoundof Example 43 in the SC_P step).LCMS Method: LCMS_AP: Rt=8.46 min, m/z m/3=1650, m/4=1237, m/5=990

UPLC Method: UPLC AP_B4_1: Rt=8.27 min Example 30N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_M; SC_M; CP_M1LCMS Method: LCMS_AP: Rt=4.52 min, m/z: m/3=1789, m/4=1342

UPLC Method: UPLC_AP_B4_1: Rt=7.63 min Example 31N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 5.Preparation method: SPPS_P; SC_P; CP_M1Maldi Method: MALDI01v1: calc. m/z: 4771.5, found m/z: 4771.6.

UPLC Method: UPLC02v1: Rt=7.5 min Example 32N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 5.Preparation method: SPPS_P; SC_P; CP_M1Maldi Method: MALDI01v1: calc. m/z: 4743.4, found m/z: 4743.3.

UPLC Method: UPLC02v1: Rt=7.2 min Example 33N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_L; SC_L; CP_M1LCMS Method: LCMS01v1: Rt=2.0 min; m/z: m/4=1358, m/5=1087

UPLC Method: UPLC02v1: Rt=7.7 min Example 34N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_L; SC_L; CP_M1LCMS Method: LCMS01v1: Rt=2.0 min; m/z: m/3=1714, m/4=1285, m/5=1029

UPLC Method: UPLC02v1: Rt=7.7 min Example 35N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(2S)-2-[[(2S)-2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(2S)-2-[[(2S)-2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_L; SC_L; CP_M1LCMS Method: LCMS01v1: Rt=2.0 min; m/z: m/3=1733, m/4=1300, m/5=1040

UPLC Method: UPLC22v1: Rt=5.83 min Example 36N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Glu²²,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 7.Preparation method: SPPS_L; SC_L; CP_M1LCMS Method: LCMS01v1: Rt=2.0 min; m/z: m/3=1632, m/4=1224, m/5=979,m/6=816

UPLC Method: UPLC02v1: Rt=7.8 min Example 37N^(ε26)-[(2S)-6-[[(2S)-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-2-(dimethylamino)hexanoyl]amino]-2-(dimethylamino)hexanoyl],N^(ε37)-[(2S)-6-[[(2S)-6-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]-2-(dimethylamino)hexanoyl]amino]-2-(dimethylamino)hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1 (using the intermediate compoundof Example 43 in the SC_P step).LCMS Method: LCMS01v1: Rt=2.50 min; m/z: m/4=1241, m/5=993, m/6=827

UPLC Method: UPLC02v1: Rt=6.5 min Example 38N^(ε26)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1LCMS Method: LCMS01v1: Rt=2 min; m/z: m/3=1986, m/4=1277, m/5=1022

UPLC Method: UPLC02v1: Rt=7.77 min Example 39N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[1-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1LCMS Method: LCMS01v: Rt=2.68 min; m/z: m/3=1704, m/4=1278

UPLC Method: UPLC02v1: Rt=8.00 min Example 40N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 3.Preparation method: SPPS_P; SC_P; CP_M1LCMS Method: LCMS01v1: Rt=2.70 min; m/z: m/3=1617, m/4=1213, m/5=971

UPLC Method: UPLC02v1: Rt=8.2 min Example 41N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Glu²²,Glu³⁰,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide

where the amino acid sequence is that of SEQ ID NO: 10.Preparation method: SPPS_P; SC_P; CP_M1LCMS Method: LCMS01v1: Rt=2.17 min; m/z: m/3=1661, m/4=1246, m/5=997

UPLC Method: UPLC02v1: Rt=8.27 min Example 42N^(ε26)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl],N^(ε37)-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib⁸,Glu²²,Glu³⁰,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptidyl-Glu

where the amino acid sequence is that of SEQ ID NO: 3.

Preparation Method: SPPS_P; SC_P; CP_M1

LCMS Method: LCMS01v1: Rt=2.18 min; m/z: m/3=1703, m/4=1278, m/5=1022

UPLC Method: UPLC02v1: Rt=8.24 min Example 43(S)-2-Dimethylamino-6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoicAcid Chem. 63

The compound of Chem. 63 is prepared as follows (further detail below):

The starting material,(S)-2-tert-Butoxycarbonylamino-6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoicacid (Novabiochem F04-12-0069; 28.0 g, 59.8 mmol), was suspended in1,4-dioxane (800 mL) and a solution of hydrogen chloride in 1,4-dioxane(7.2 M, 500 mL) was added and the resulting suspension was stirred atroom temperature overnight. Then it was filtered and thoroughly washedwith 1,4-dioxane and diethylether prior to drying in vacuo. Thehydrochloride of(S)-2-amino-6-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid wasobtained as a white solid.

Yield: 23.58 g (96%)

NMR: 1H NMR spectrum (300 MHz, MeOD-d4, dH): 7.80 (d, J=7.4 Hz, 2H);7.64 (d, J=7.4 Hz, 2H); 7.47-7.25 (m, 4H); 4.58-4.29 (m, 2H); 4.14-4.25(m, 1H); 3.95 (t, J=6.3 Hz, 1H); 3.19-2.73 (m, 2H); 2.09-1.71 (m, 2H);1.65-1.14 (m, 4H)

The product from the above reaction (13.1 g, 32.4 mmol) and an aqueoussolution of formaldehyde (approx. 35%, 13.2 mL) were dissolved in amixture of 1,4-dioxane/methanol (300 mL, 1:1) and the resulting solutionwas cooled down to 0° C. Then sodium borohydride (5.50 g, 145 mmol) wascarefully added in three portions within 15 minutes and pH of thereaction mixture was adjusted by addition of acetic acid (14.4 mL) to pH5.5. Then a second portion of aqueous formaldehyde (approx. 35%, 13.2mL) was added into the reaction mixture and another part of sodiumborohydride (5.50 g, 145 mmol) was carefully added in three portions.The reaction mixture was allowed to warm up to the room temperature andstirred for further 60 minutes. HPLC-MS analysis revealed fullconversion of the starting material thus the reaction was quenched byaddition of 10% aqueous sodium hydrogencarbonate solution (approx. 20mL) until pH 7.0. Then the reaction mixture was diluted with saturatedaqueous solution of sodium chloride and extracted with chloroform (5×500mL). Fractions containing the desired product (TLC) were collected anddried with anhydrous magnesium sulfate. Then the solution was filteredand the solvents removed under reduced pressure. The crude product wasdissolved in warm chloroform (300 mL) and a mixture ofdiethylether/hexane (2:1, 700 mL) was added. The formed precipitate wasfiltered, washed with diethylether prior to drying in vacuo to give thetitle compound as a white powder.

Yield: 14.26 g

LC-MS (Sunfire 4.6 mm×100 mm, acetonitrile/water 35:65 to 100:0+0.1%FA): Rt=3.71 min

LC-MS m/z: 397.4 (M+H)+.

The collected crude product (36.5 g) from 3 batches of the above wassubjected to RP-column chromatography (Cromasil C18, 100 Å, 13 μm,7.5×41 cm, flow rate 200 mL/min, acetonitrile/water 20:80 to 45:55,detection UV 220 nm). Fractions containing the product were collectedand freeze dried.(S)-2-Dimethylamino-6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoicacid was obtained as fine white powder.

Yield: 20.21 g

NMR: 1H NMR spectrum (300 MHz, AcOD-d4, dH): 7.87-7.72 (m, 2H);7.70-7.57 (m, 2H); 7.53-7.20 (m, 4H); 4.66-4.36 (m, 2H); 4.35-4.12 (m,1H); 3.98-3.77 (m, 1H); 3.32-3.10 (m, 2H); 2.95 (s, 6H); 2.16-1.73 (m,2H, overlapped); 1.69-1.16 (m, 4H)

LC-MS (Sunfire 4.6 mm×100 mm, acetonitrile/water 35:65 to 100:0+0.1%FA): Rt=3.11 min

LC-MS m/z: 397.2 (M+H)+

This intermediate product may be used in the synthesis of the compoundsof Examples 29 and 37 and other compounds with the same linker.

Pharmacological Methods Example 44: In Vitro Potency(AlphaScreen—Membranes)

The purpose of this example is to test the activity, or potency, of theGLP-1 derivatives in vitro.

The potencies of the GLP-1 derivatives of Examples 1-21 and 23 weredetermined as described below, i.e. as the stimulation of the formationof cyclic AMP (cAMP) in a medium containing membranes expressing thehuman GLP-1 receptor.

Principle

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor werestimulated with the GLP-1 analogue or derivative in question, and thepotency of cAMP production was measured using the AlphaScreen™ cAMPAssay Kit from Perkin Elmer Life Sciences. The basic principle of theAlphaScreen Assay is a competition between endogenous cAMP andexogenously added biotin-cAMP. The capture of cAMP is achieved by usinga specific antibody conjugated to acceptor beads.

Cell Culture and Preparation of Membranes

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 5% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the selectionmarker G418.

Cells at approximately 80% confluence were washed 2× with PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and thesupernatant removed. The additional steps were all performed on ice. Thecell pellet was homogenised by the Ultrathurax for 20-30 s in 10 ml ofBuffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at20,000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mMNa-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenised for 20-30s and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,homogenisation and centrifugation was repeated once and the membraneswere resuspended in Buffer 2. The protein concentration was determinedand the membranes stored at −80° C. until use.

The assay was performed in flat-bottom 96-well plates (Costar cat.no:3693). The final volume per well was 50 μl.

Solutions and Reagents

AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No:6760625M); containing Anti-cAMP Acceptor beads (10 U/μl), StreptavidinDonor beads (10 U/μl) and Biotinylated-cAMP (133 U/μl).

AlphaScreen Buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat. no: T3253); 5 mMHEPES (Sigma, cat. no: H3375); 10 mM MgCl₂, 6H₂O (Merck, cat. no: 5833);150 mM NaCl (Sigma, cat. no: S9625); 0.01% Tween (Merck, cat. no:822184). The following was added to the AlphaScreen Buffer prior to use(final concentrations indicated): BSA (Sigma, cat. no. A7906): 0.1%;IBMX (Sigma, cat. no. 15879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM;GTP (Sigma, cat. no. G8877): 1 uM.

cAMP standard (dilution factor in assay=5): cAMP Solution: 5 μL of a 5mM cAMP-stock+495 μL AlphaScreen Buffer.

Suitable dilution series in AlphaScreen Buffer were prepared of the cAMPstandard as well as the GLP-1 analogue or derivative to be tested, e.g.the following eight concentrations of the GLP-1 compound: 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ and 10⁻¹⁴M, and a series from, e.g.,10⁻⁶ to 3×10⁻¹¹ of cAMP.

Membrane/Acceptor Beads

Membranes were prepared from hGLP-1/BHK 467-12A cells with aconcentration of 6 μg/well corresponding to 0.6 mg/ml (the amount ofmembranes used pr. well may vary)

“No membranes”: Acceptor Beads (15 μg/ml final) in AlphaScreen buffer

“6 μg/well membranes”: membranes+Acceptor Beads (15 μg/ml final) inAlphaScreen buffer

An aliquot (10 μl) of “No membranes” was added to the cAMP standard (perwell in duplicate wells) and the positive and negative controls

An aliquot (10 μl) of “6 μg/well membranes” was added to GLP-1 andanalogues (per well in duplicate or triplicate wells)

Pos. Control: 10 μl “no membranes”+10 μl AlphaScreen Buffer

Neg. Control: 10 μl “no membranes”+10 μl cAMP Stock Solution (50 μM)

As the beads are sensitive to direct light, any handling was in the dark(as dark as possible), or in green light. All dilutions were made onice.

Procedure 1. Make the AlphaScreen Buffer.

2. Dissolve and dilute the GLP-1/Analogues/cAMP standard in AlphaScreenBuffer.3. Make the Donor Beads Solution by mixing streptavidin donor beads (2units/well) and biotinylated cAMP (1.2 units/well) and incubate 20-30min in the dark at room temperature4. Add the cAMP/GLP-1/Analogues to the plate: 10 μl per well.5. Prepare membrane/Acceptor Beads solution and add this to the plates:10 μl per well.6. Add the Donor Beads: 30 μl per well.7. Wrap the plate in aluminum foil and incubate on the shaker for 3hours (very slowly) at RT.8. Count on AlphaScreen—each plate pre incubates in the AlphaScreen for3 minutes before counting.

Results

The EC₅₀ [pM] values were calculated using the Graph-Pad Prism software(version 5) and are shown in Table 1 below. The potency of allderivatives in vitro was confirmed.

TABLE 1 In vitro potency (AlphaScreen) Compound of Example no. EC₅₀/pM 159 2 44 3 48 4 83 5 33 6 634 7 136 8 34 9 116 10 492 11 426 12 227 13385 14 280 15 155 16 154 17 286 18 75 19 57 20 71 21 369 23 252 26 13527 3685 28 7250

All derivatives except two had a good in vitro potency corresponding toan EC₅₀ of below 1200 pM. For comparison, compound no. 13 in Table 1 ofJournal of Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-669(GLP-1(7-37) acylated at K^(26,34) with bis-C12-diacid) had an in vitropotency corresponding to an EC₅₀ of 1200 pM.

Example 45: In Vitro Potency (CRE Luciferase; Whole Cells)

The purpose of this example is to test the activity, or potency, of theGLP-1 derivatives in vitro. The in vitro potency is the measure of humanGLP-1 receptor activation in a whole cell assay.

The potencies of the GLP-1 derivatives of Examples 1-42 were determinedas described below. Semaglutide was included for comparison.

Principle

In vitro potency was determined by measuring the response of human GLP-1receptor in a reporter gene assay. The assay was performed in a stablytransfected BHK cell line that expresses the human GLP-1 receptor andcontains the DNA for the cAMP response element (CRE) coupled to apromoter and the gene for firefly luciferase (CRE luciferase). When thehuman GLP-1 receptor was activated it results in the production of cAMP,which in turn results in the luciferase protein being expressed. Whenassay incubation was completed the luciferase substrate (luciferin) wasadded and the enzyme converts luciferin to oxyluciferin and producesbioluminescence. The luminescence was measured and was the readout forthe assay.

In order to test the binding of the derivatives to albumin, the assaywas performed in the absence of serum albumin as well as in the presenceof a considerably higher concentration of serum albumin (1.0% finalassay concentration). An increase of the in vitro potency, EC₅₀ value,in the presence of serum albumin indicates an affinity to serum albuminand represents a method to predict a protracted pharmacokinetic profileof the test substance in animal models.

Cell Culture and Preparation

The cells used in this assay (clone FCW467-12A/KZ10-1) were BHK cellswith BHKTS13 as a parent cell line. The cells were derived from a clone(FCW467-12A) that expresses the human GLP-1 receptor and wereestablished by further transfection with CRE luciferase to obtain thecurrent clone.

The cells were cultured at 5% CO₂ in cell culture medium. They werealiquoted and stored in liquid nitrogen. Before each assay an aliquotwas taken up and washed twice in PBS before being suspended at thedesired concentration in the assay specific buffer. For 96-well platesthe suspension was made to give a final concentration of 5×10³cells/well.

Materials

The following chemicals were used in the assay: Pluronic F-68 (10%)(Gibco 2404), human serum albumin (HSA) (Sigma A9511), ovalbumin (SigmaA5503), DMEM w/o phenol red (Gibco 11880-028), 1 M Hepes (Gibco 15630),Glutamax 100× (Gibco 35050) and steadylite plus (PerkinElmer 6016757).

Buffers

Cell culture medium consisted of 10% FBS (Fetal Bovine Serum), 1 mg/mlG418, 240 nM MTX (methotrexate) and 1% pen/strep(penicillin/streptomycin. Assay medium consisted of DMEM w/o phenol red,10 mM Hepes and 1× Glutamax. The 1% assay buffer consisted of 2%ovalbumin, 0.2% Pluronic F-68 and 2% HSA in assay medium. The 0% assaybuffer consisted of 2% ovalbumin and 0.2% Pluronic F-68 in assay medium.

Procedure

1) Cell stocks were thawed in a 37° C. water bath.2) Cells were washed three times in PBS.3) The cells were counted and adjusted to 5×10³ cells/50 μl (1×10⁵cells/ml) in assay medium. A 50 μl aliquot of cells was transferred toeach well in the assay plate.4) Stocks of the test compounds and reference compounds were diluted toa concentration of 0.2 μM in 0% assay buffer for the 0% HSA CREluciferase assay and 1% assay buffer for the HSA CRE luciferase assay.Compounds were diluted 10-fold to give the following concentrations:2×10⁻⁷ M, 2×10⁻⁸ M; 2×10⁻⁹ M, 2×10⁻¹⁰ M, 2×10⁻¹¹ M, 2×10⁻¹² M and2×10⁻¹³ M. For each compound a blank assay buffer control was alsoincluded.5) A 50 μl aliquot of compound or blank was transferred in triplicatefrom the dilution plate to the assay plate. Compounds were tested at thefollowing final concentrations: 1×10⁻⁷ M, 1×10⁻⁸ M; 1×10⁻⁹ M, 1×10⁻¹⁰ M,1×10⁻¹¹ M, 1×10⁻¹² M and 1×10⁻¹³ M.6) The assay plate was incubated for 3 h in a 5% CO₂ incubator at 37° C.7) The assay plate was removed from the incubator and allowed to standat room temperature for 15 min.8) A 100 μl aliquot of steadylite plus reagent was added to each well ofthe assay plate (reagent was light sensitive).9) Each assay plate was covered with aluminum foil to protect it fromlight and shaken for 30 min at room temperature.10) Each assay plate was read in a Packard TopCount NXT instrument.

Calculations and Results

The data from the TopCount instrument were transferred to GraphPad Prismsoftware. The software averages the values for each replicate andperforms a non-linear regression. EC₅₀ values were calculated by thesoftware and are shown in Table 2 below (in pM).

TABLE 2 In vitro potency (CRE luciferase) Compound of EC₅₀/pM EC₅₀/pMEC₅₀/pM Example no. (0% HSA) (1% HSA) (ratio 1% HSA/0% HSA) 1 4.0 57 152 6.2 59 9.5 3 7.8 80 10.3 4 6.8 1615 307 5 5.4 33 6.0 6 3.3 4570 1508 75.8 96 16 8 2.6 45 24 9 8.7 633 72 10 8.0 3630 456 11 7.3 1570 217 125.5 1147 194 13 2.2 1384 659 14 4.8 76 16 15 3.7 327 93 16 3.3 47 14 173.3 206 62 18 12 79 6.9 19 25 51 2.5 20 4.9 63 13 21 3.6 141 39 22 5.570 13 23 4.6 156 34 24 5.0 126 37 25 9.4 124 19 26 5.9 231 39 27 22 6190276 28 17 5730 329 29 7.9 427 54 30 5.4 93 17 31 7.2 550 82 32 11 162 1433 7.4 69 11 34 7.9 59 9.1 35 5.5 108 20 36 4.9 65 14 37 6.3 93 15 387.6 189 26 39 3.7 140 38 40 2.6 213 84 41 1.5 391 260 42 1.4 354 251

All derivatives had a good in vitro potency corresponding to an EC₅₀ at0% HSA of below 200 pM.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1 (7-37) acylated atK^(26,34) with bis-C12-diacid) had an in vitro potency corresponding toan EC₅₀ at 0% HSA of 440 pM, an EC₅₀ at 1% HSA of 3317 pM, and a ratio(1% HSA/0% HSA) of 7.5.

Example 46: GLP-1 Receptor Binding

The purpose of this experiment is to investigate the binding to theGLP-1 receptor of the GLP-1 derivatives, and how the binding ispotentially influenced by the presence of albumin. This is done in an invitro experiment as described below.

The binding affinity of the GLP-1 derivatives of Examples 1-43 to thehuman GLP-1 receptor was measured by way of their ability to displace of¹²⁵I-GLP-1 from the receptor. In order to test the binding of thederivatives to albumin, the assay was performed with a low concentrationof albumin (0.001%—corresponding to the residual amount thereof in thetracer), as well as with a high concentration of albumin (2.0% added). Ashift in the binding affinity, IC₅₀, is an indication that the peptidein question binds to albumin, and thereby a prediction of a potentialprotracted pharmacokinetic profile of the peptide in question in animalmodels.

Conditions

Species (in vitro): Hamster

Biological End Point: Receptor Binding

Assay Method: SPA

Receptor: GLP-1 receptor

Cell Line: BHK tk-ts13

Cell Culture and Membrane Purification

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 10% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418.

The cells (approx. 80% confluence) were washed twice in PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), following which they were separated bycentrifugation at 1000 rpm for 5 min. The cells/cell pellet must be kepton ice to the extent possible in the subsequent steps. The cell pelletwas homogenised with Ultrathurrax for 20-30 seconds in a suitable amountof Buffer 1 (depending on the amount of cells, but e.g. 10 ml). Thehomogenate was centrifuged at 20000 rpm for 15 minutes. The pellet wasresuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. Thisstep was repeated once more. The resulting pellet was resuspended inBuffer 2, and the protein concentration was determined. The membraneswere stored at minus 80° C.

Buffer 1: 20 mM Na-HEPES+10 mM EDTA, pH 7.4

Buffer 2: 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4

Binding Assay:

SPA:

Test compounds, membranes, SPA-particles and [¹²⁵I]-GLP-1 (7-36)NH₂ werediluted in assay buffer. 50 ul (micro liter) HSA (“high albumin”experiment containing 2% HSA), or buffer (“low albumin” experimentcontaining 0.001% HSA) was added to Optiplate, and 25 ul of testcompounds were added. 5-10 ug membrane protein/sample was added (50 ul)corresponding to 0.1-0.2 mg protein/ml (to be preferably optimised foreach membrane preparation). SPA-particles (Wheatgerm agglutinin SPAbeads, Perkin Elmer, #RPNQ0001) were added in an amount of 0.5 mg/well(50 ul). The incubation was started with [¹²⁵I]-GLP-1]-(7-36)NH₂ (finalconcentration 0.06 nM corresponding to 49.880 DPM, 25 ul). The plateswere sealed with PlateSealer and incubated for 120 minutes at 30° C.while shaking. The plates were centrifuged (1500 rpm, 10 min) andcounted in Topcounter.

Assay Buffer:

50 mM HEPES

5 mM EGTA

5 mM MgCl₂

0.005% Tween 20

pH 7.4

HSA was SIGMA A1653

Calculations

The IC₅₀ value was read from the curve as the concentration whichdisplaces 50% of ¹²⁵I-GLP-1 from the receptor.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible (good potency),corresponding to a low IC₅₀ value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their lifetime in plasma.Therefore, the IC₅₀ value at high albumin will generally be higher thanthe IC₅₀ value at low albumin, corresponding to a reduced binding to theGLP-1 receptor, caused by albumin binding competing with the binding tothe GLP-1 receptor.

Results

The following results were obtained:

TABLE 3 Receptor binding affinity Compound of Example no. IC₅₀/nM (lowHSA) IC₅₀/nM (high HSA) 1 0.22 13 2 0.24 29 3 0.93 71 4 0.48 172 5 1.0364 6 0.32 ≥1000 7 0.13 106 8 0.68 269 9 0.64 321 10 0.84 490 11 0.74 52212 0.33 222 13 0.76 545 14 0.30 154 15 0.35 303 16 0.24 199 17 0.42 63218 0.99 159 19 0.77 147 20 0.13 48 21 0.22 484 22 0.26 251 23 0.27 10224 0.27 136 25 0.18 274 26 0.38 106 27 0.25 689 28 1.67 ≥1000 29 0.55 5630 0.42 203 31 0.76 40 32 1.57 27 33 0.31 33 34 0.12 45 35 0.08 36 360.10 25 37 0.11 24 38 0.21 53 39 0.06 131 40 0.03 195 41 0.03 641 420.02 123

All derivatives had an IC₅₀ (low albumin) below 2.0 nM. As regards IC₅₀(high albumin), with two exceptions all derivatives had an IC₅₀ (highalbumin) below 1000 nM.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1 (7-37) acylated atK^(26,34) with bis-C12-diacid) had an IC₅₀ (low albumin) of 17.7 nM, andan IC₅₀ (high albumin) of 908 nM.

Example 47: Pharmacokinetic (PK) Study in Minipig

The purpose of this study is to determine the protraction in vivo of theGLP-1 derivatives after i.v. administration to minipigs, i.e. theprolongation of their time of action. This is done in a pharmacokinetic(PK) study, where the terminal half-life of the derivative in questionis determined. By terminal half-life is generally meant the period oftime it takes to halve a certain plasma concentration, measured afterthe initial distribution phase.

The derivatives of Examples 6, 7, 10, 11, 12, 13, 14, 18, and 22 weresubjected to PK study A (see below), whereas the derivatives of Examples9, 16, 17, 19, 20, 24, 26, 29, and 36 were subjected to PK study B (seebelow).

Study A: Male Göttingen minipigs were obtained from Ellegaard GöttingenMinipigs (Dalmose, Denmark) approximately 7-14 months of age andweighing from approximately 16-35 kg were used in the studies. Theminipigs were housed individually and fed restrictedly once or twicedaily with SDS minipig diet (Special Diets Services, Essex, UK). Afterat least 2 weeks of acclimatisation two permanent central venouscatheters were implanted in vena cava caudalis or cranialis in eachanimal. The animals were allowed 1 week recovery after the surgery, andwere then used for repeated pharmacokinetic studies with a suitablewash-out period between successive GLP-1 derivative dosings.

The animals were fasted for approximately 18 h before dosing and from 0to 4 h after dosing, but had ad libitum access to water during the wholeperiod.

The GLP-1 derivatives were dissolved in 50 mM sodium phosphate, 145 mMsodium chloride, 0.05% tween 80, pH 7.4 to a concentration of usuallyfrom 20-60 nmol/ml. Intravenous injections (the volume corresponding tousually 1-2 nmol/kg, for example 0.033 ml/kg) of the compounds weregiven through one catheter, and blood was sampled at predefined timepoints for up till 13 days post dosing (preferably through the othercatheter). Blood samples (for example 0.8 ml) were collected in EDTAbuffer (8 mM) and then centrifuged at 4° C. and 1942G for 10 minutes.

Study B:

Male Göttingen minipigs were obtained from Ellegaard Göttingen Minipigs(Dalmose, Denmark) approximately 5 months of age and weighing fromapproximately 9 kg were used in the studies. The minipigs were housed inpens with straw as bedding, six together in each pen and fedrestrictedly once or twice daily with Altromin 9023 minipig diet (Chr.Petersen A/S, DK-4100 Ringsted) or Standard SMP(E) minipig diet (SpecialDiet Services (SDS), UK). The pigs were used for repeatedpharmacokinetic studies with a suitable wash-out period betweensuccessive GLP-1 derivative dosings. An acclimatisation period of 1 to 4weeks was allowed during which time the minipigs was trained to befixated on the backs for blood sampling and in slings for i.v. dosing.All handling, dosing and blood sampling of the animals will be performedby trained and skilled staff.

The animals were fasted for approximately 18 h before dosing and from 0to 4 h after dosing, but had ad libitum access to water during the wholeperiod.

The GLP-1 derivatives were dissolved in 50 mM sodium phosphate, 145 mMsodium chloride, 0.05% tween 80, pH 7.4 to a concentration of usuallyfrom 20-60 nmol/ml. Intravenous injections (the volume corresponding tousually 2 nmol/kg, for example 0.1 ml/kg) of the compounds were given asintravenous injections via a Venflon inserted in an ear vein, while theyare placed unanaesthetised in a sling. The dose volume was 0.1 ml/kg,and blood was sampled at predefined time points for up till 17 days postdosing (samples was taken with syringe from a jugular vein). Bloodsamples (for example 0.8 ml) were collected in EDTA buffer (8 mM) andthen centrifuged at 4° C. and 2000G for 10 minutes.

Sampling and Analysis (Study A and B):

Plasma was pippetted into Micronic tubes on dry ice, and kept at −20° C.until analyzed for plasma concentration of the respective GLP-1 compoundusing ELISA or a similar antibody based assay or LC-MS. Individualplasma concentration-time profiles were analyzed by a non-compartmentalmodel in Phoenix WinNonLin ver. 6.2. (Pharsight Inc., Mountain View,Calif., USA), and the resulting terminal half-lives (harmonic mean)determined.

Results

A number of Example compounds were tested, and the results are shown inTable 4, below.

TABLE 4 Half-life in minipigs Compound of Example no. Minipig iv PK, T½(hours) 6 98 7 69 9 72 10 97 11 95 12 109 13 93 14 88 16 64 17 71 18 4719 56 20 59 22 59 24 123 26 13 29 82 36 69

All compounds tested had a half-life well above 5 hours, and mostcompounds had a very fine half-life of above 50 hours.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1 (7-37) acylated atK^(26,34) with bis-C12-diacid) had a half-life of 5 hours.

Example 48: Pharmacokinetic (PK) Study in Rat

The purpose of this Example is to investigate half-life in vivo in rat.

In vivo pharmacokinetic studies in rats were performed with the GLP-1derivatives of a number of the Example compounds, as described in thefollowing. Semaglutide was included for comparison.

Male Sprague Dawley rats of same age with a body weight of approximately400 g were obtained from Taconic (Denmark) and assigned to thetreatments by simple randomisation on body weight, approximately 4 ratsper group.

The GLP-1 derivatives (approximately 6 nmol/ml) were dissolved in 50 mMsodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4.Intravenous injections (1.0 ml/kg) of the compounds were given with asyringe in the tail vein of conscious rats. Blood was sampled from venasublingualis for 5 days post dosing. Blood samples (200 μl) werecollected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 10000Gfor 5 minutes. Plasma samples were kept at −20° C. until analyzed forplasma concentration of the respective GLP-1 compound.

The plasma concentrations of the GLP-1 compounds were determined using aLuminescence Oxygen Channeling Immunoasssay (LOCI), generally asdescribed for the determination of insulin by Poulsen and Jensen inJournal of Biomolecular Screening 2007, vol. 12, p. 240-247. The donorbeads were coated with streptavidin, while acceptor beads wereconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, was biotinylated. The three reactants were combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which werechanneled into the acceptor beads and triggered chemiluminescence whichwas measured in an Envision plate reader. The amount of light wasproportional to the concentration of the compound.

Plasma concentration-time profiles were analyzed using Phoenix WinNonLinver. 6.2, Pharsight Inc., Mountain View, Calif., USA), and the half-life(T_(1/2)) calculated using individual plasma concentration-time profilesfrom each animal.

Results

The results are shown in Table 5, below.

TABLE 5 Half-life in rat Compound of Example no. PK in rat T½ (hours) 114 2 17 4 17 6 25 7 17 8 14 9 20 10 28 11 28 12 28 13 31 14 19 15 20 1617 17 24 18 20 19 13 20 14 22 15 23 19 26 16 27 21 29 19 30 16

All tested compounds had a half-life of at least 13 hours. The half-lifeof semaglutide tested in the same set-up but with n=8 was 11 hours.

Example 49: Pharmacodynamic (PD) Study in Pigs

The purpose of this experiment was to investigate the effect of a coupleof Example compounds on food intake in pigs. This was done in apharmacodynamic (PD) study as described below, in which food intake wasmeasured from 1 to 4 days after administration of a single dose of theGLP-1 derivative, as compared to a vehicle-treated control group.

Female Landrace Yorkshire Duroc (LYD) pigs, approximately 3 months ofage, weighing approximately 30-35 kg were used (n=3-4 per group). Theanimals were housed in a group for approximately 1 week duringacclimatisation to the animal facilities. During the experimental periodthe animals were placed in individual pens at least 2 days before dosingand during the entire experiment for measurement of individual foodintake. The animals were fed ad libitum with pig fodder (SvinefoderDanish Top) at all times both during the acclimatisation and theexperimental period. Food intake was monitored on line by logging theweight of fodder every 15 minutes. The system used was Mpigwin(Ellegaard Systems, Faaborg, Denmark).

The GLP-1 derivatives were dissolved in a phosphate buffer (50 mM sodiumphosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4) atconcentrations of 12, 40, 120, 400 or 1200 nmol/ml corresponding todoses of 0.3, 1, 3, 10 or 30 nmol/kg. The phosphate buffer serves asvehicle. Animals are dosed with a single subcutaneous dose of the GLP-1derivative or vehicle (dose volume 0.025 ml/kg) on the morning of day 1,and food intake is measured for 1 day after dosing. On the last day ofeach study, 1-4 days after dosing, a blood sample for measurement ofplasma exposure of the GLP-1 derivative is taken from the heart inanaesthetised animals. The animals are thereafter euthanised with anintra-cardial overdose of pentobarbitone. Plasma content of the GLP-1derivatives is analysed using ELISA or a similar antibody based assay orLC-MS.

Food intake is calculated as mean±SEM food intake in 24 h intervals(0-24 h, 24-48 h, and 48-72 h). In Table 6 below the food intake isindicated as percentage of the food intake of the vehicle group in thesame time interval (dosage 3.0 nmol/kg).

Statistical comparisons of the food intake in the 24 h intervals in thevehicle vs. GLP-1 derivative group are done using two-way-ANOVA repeatedmeasures, followed by Bonferroni post-test.

Results

The results are shown in Table 6, below.

TABLE 6 Effect on food intake in pigs PD in pig, food intake (% ofvehicle) for hours (x-y) Compound of Time interval (h) Example no. 0-2424-48 48-72 6 84 85 73 7 14 18 10 105 104 16 33 43 19 52 64 24 73 73 2610 26 52

All but one of these compounds showed a very nice reduction in foodintake. This is so in particular for the compound of Examples 7 and 26.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1 (7-37) acylated atK^(26,34) with bis-C12-diacid) when tested in the same way had a foodintake (% of vehicle) for hours 0-24, 24-48, and 48-72, of 95%, 96%, and103%, respectively.

Example 50: Comparative Example—In Vitro Potency and GLP-1 ReceptorBinding

The purpose of this Example is to compare the in vitro potency and GLP-1receptor binding of those GLP-1 derivatives of the invention for which adirect comparator compound differing only in the linker part of thederivative is found in the prior art.

The direct comparator compounds are all disclosed in WO2011/080103.

Table 7 below shows the Example nos. of the compounds in question, aswell as their structure in outline, and Table 8 shows their in vitropotency and GLP-1 receptor binding data, determined as described inExamples 44, 45, and 46 herein.

The abbreviations used in Table 7 to characterise protracting moietiesand linkers are defined as follows: C16 diacid refers to Chem. 1 withx=14; C14 diacid refers to Chem. 1 with x=12; 4-COOH-PhO-C10 refers toChem. 2 with γ=9; gGlu refers to Chem. 14; eps-Lys refers to Chem. 3with w=0 and q=4 (Chem. 6); and OEG refers to Chem. 12 with k=n=1 (Chem.13).

There are four sets of derivatives of the invention versus their directcomparator compound known from WO2011/080103, and these sets areseparated by double lines in the below Tables 7 and 8.

TABLE 7 Structure of compounds and comparative compounds Comparativecompound Amino acid Derivative of (WO 2011/ changes (rel. to AcylationProtracting the invention 080103) SEQ ID NO: 1) positions moiety LinkerExample 6 8Aib, 34R, 37K 26K, 37K C16 diacid gGlu-2xeps-Lys Example 108Aib, 34R, 37K 26K, 37K C16 diacid gGlu-2xOEG-eps-Lys Example 11 8Aib,34R, 37K 26K, 37K C16 diacid 2xgGlu-OEG-eps-Lys Example 12 8Aib, 34R,37K 26K, 37K C16 diacid 2xgGlu-2xeps-Lys Example 13 8Aib, 34R, 37K 26K,37K C16 diacid 3xgGlu-eps-Lys Example 3 8Aib, 34R, 37K 26K, 37K C16diacid gGlu-2xOEG Example 2 8Aib, 34R, 37K 26K, 37K C14 diacidgGlu-2xeps-Lys Example 19 8Aib, 34R, 37K 26K, 37K C14 diacidgGlu-2xOEG-eps-Lys Example 4 8Aib, 34R, 37K 26K, 37K C14 diacidgGlu-2xOEG Example 1 8Aib, 34R, 37K 26K, 37K 4-COOH—PhO—C10gGlu-2xeps-Lys Example 5 8Aib, 34R, 37K 26K, 37K 4-COOH—PhO—C10gGlu-2xOEG-eps-Lys Example 2 8Aib, 34R, 37K 26K, 37K 4-COOH—PhO—C10gGlu-2xOEG Example 32 8Aib, 31H, 34Q, 37K 26K, 37K 4-COOH—PhO—C10gGlu-2xeps-Lys Example 17 8Aib, 31H, 34Q, 37K 26K, 37K 4-COOH—PhO—C10gGlu-2xOEG

TABLE 8 Comparison of in vitro potency and GLP-1 receptor binding dataGLP-1 GLP-1 In vitro potency In vitro potency receptor binding receptorbinding Comparative In vitro potency (Example 45) (Example 45) (Example46) (Example 46) Derivative of compound of (Example 44) (0% HSA) (1%HSA) (low HSA) (high HSA) the invention WO 2011/080103 EC50/pM EC50/pMEC50/pM IC50/nM IC50/nM Example 6 634 3.3 4570 0.32 ≥1000 Example 10 4928.0 3630 0.84 490 Example 11 426 7.3 1570 0.74 522 Example 12 227 5.51147 0.33 222 Example 13 385 2.2 1384 0.76 545 Example 3 1683 41 49103.55 751 Example 2 44 6.2 59 0.24 29 Example 19 57 25 51 0.77 147Example 4 366 46 92 6.02 357 Example 1 59 4.0 57 0.22 13 Example 5 335.4 33 1.03 64 Example 2 112 6.7 221 2.27 485 Example 32 — 11 162 1.5727 Example 17 582 132 356 45 ≥1000

The results in Table 8 show that the derivatives of the invention aremuch more potent and bind much better to the GLP-1 receptor as comparedto their respective direct comparator compound known from WO2011/080103which differs only in the linker.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A compound selected from the group consisting of

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 7,

wherein the amino acid sequence is that of SEQ ID NO: 9,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 5,

wherein the amino acid sequence is that of SEQ ID NO: 5,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 7,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 3,

wherein the amino acid sequence is that of SEQ ID NO: 10, and

wherein the amino acid sequence is that of SEQ ID NO: 3.